Development of the chromatogram

Development of the Chromatogram. The term development describes the process of performing a chromatographic separation. There are several ways in which separation may be made to occur, eg, frontal, displacement, and elution chromatography. Frontal chromatography uses a large quantity of sample and is usually unsuited to analytical procedures. In displacement and elution chromatography, much smaller amounts of material are used. 

Every chromatographic investigation begins with the preparation of the sample and the chromatographic system. This is followed by the crux of the separation process (development of the chromatogram) which is in turn followed by the visualization of the separated substances and the preservation of the chromatogram and finally by the analysis of the results. 

N,N -Diacetylkasuganobiosamine. To a suspension of kasuganobiosamine (500 mg., 1.6 mmoles) in 10 ml. of methanol was added 2.5 ml. of acetic anhydride. The mixture became a colorless transparent solution after allowing to stand at room temperature for 5 hours. After adding 20 ml. of water, the reaction mixture was allowed to stand at room temperature for 2 hours. An oily material was obtained after removal of the solvent, and dissolved in 50 ml. of water to adjust to pH 5.0 with dilute sodium hydroxide. The solution was placed and passed on a column (1.5 x 20 cm.) of Amberlite CG-50 (H form). Development of the chromatogram with water afforded fractions positive to Lemieux reaction. A colorless powder (620 mg.) was obtained after removal of the solvent. The powder was dissolved in methanol (15 ml.) to remove insoluble material. To the filtrate was added ether until no precipitate was produced. This procedure was repeated twice, affording a colorless material, m.p. 143°-150°C., [ ]D20 +113° (c=l.l, H20) which was identified to be 2V,N -diacetyl derivative of 4. The yield was 313 mg. (0.79 mmole). Anal. Calcd. for C16H2809N2 C, 48.97 H, 7.19 O, 36.70 N, 7.14. Found C, 48.93 H, 7.46 O, 37.10 N, 6.92. 

Further development of the chromatogram with 0.1N ammonia afforded fractions positive to ninhydrin test. From the fractions, 249 mg. of a colorless material was obtained. It was dissolved in 19 ml. of water and the solution was adjusted to pH 4.0 with dilute hydrochloric acid. A colorless material, after condensing under a reduced pressure and lyophilization, was recrystallized from aqueous methanol with a small amount... 

It is quite obvious that dispersion in the direction y (perpendicular to the direction of the development of the chromatogram) cannot change the shape of the longitudinal cross-section of the chromatographic spots. Because of the qualitative nature of our discussion, the dispersion in the direction y was ignored. 

Figure 6.27c, this procedure is illustrated by showing the band of the mixture throughout the whole width of the chromatographic plate [26,27]. In the next stage, the development of the chromatogram proceeds. It is performed by the introduction of an appropriate developing solvent into the eluent reservoir. It is recommended that at hrst a small quantity of the eluent be introduced into the reservoir to wash the reservoir walls and eliminate remnants of the sample solution. An example of separation of the sample mixture using this procedure is shown in Figure 6.28. Even a few milliliters of the sample solution can be applied with this mode. However, it depends on the sample mixture.

Figure 7.42 Appearance of chromatographic spots, (a) After HPLC separation and deposition on the TLC plate (b) after development of the chromatogram with benzene. After Fujimoto et al. [931], Reprinted from Journal of Chromatography, 438, C. Fujimoto et al., 329-337, Copyright (1988), with permission from Elsevier...

Coloured substances can be observed visually in situ after development of the chromatogram, but those which are colourless must be visualized by physical or chemical means. For many samples, and especially for... 

Figure 13.9. Thin layer at start (left) and after development (right). Right chromatogram shows the distance the eluent and spots moved during the development of the chromatogram.

The paper chromatograms were developed on Whatman filter paper No. 1, chromatography grade, using sheets 6" x 22 " in size, with development of the chromatograms in the machine direction of the paper. 

Your laboratory period had only 90 min. for the development of the chromatogram. In order to get better separation of the spots you must allow the solvent front to move much farther than the value you reported on your Report Sheet. Assuming a steady rate of solvent movement, how long of a lab period do you need for the solvent front to move 12.5 cm ... 

Equilibrate the atmosphere in the Developing Chamber by placing in it a volume of the mobile phase in excess of that required for complete development of the chromatogram, cover the chamber with its lid, and allow it to stand for at least 30 min. 

They are passed into the column for the development of the chromatogram. In this process, the various zones of the chromatogram get separated sharply from one another. The solvents when used for this purpose are termed as developers. The developer is generally a solvent in which the components of a mixture are not highly soluble. It is usually a liquid of low molar mass. Attempt is often made to use the same solvent which is used for introducing the mixture, as a developer. But it is not always possible. Hence, other suitable solvents are used quite often. 

The isolation of the individual chains is usually made with a chromatographic method pioneered by Clegg et al. (CIS). Modifications of the method have been made to suit specific purposes in the isolation of the chains of both animal and human hemoglobin. Type of resin, size of column, pH, and molarity of developers have been adjusted or changed for development of the chromatogram desired (A2). 

The efficiency of separation in all types of column chromatography depends on the extent to which the sample bandwidth broadens during development of the chromatogram. Three factors determine this broadening (1) axial diffusion of solutes within the mobile phase, which is... 

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 remaining water layer is extracted six times with 10-ml portions of n-butanol ethyl acetate (1 1) and the combined organic solutions are evaporated to dryness under vacuum. The residue is dissolved in 1 ml of ethyl acetate acetic acid n-butanol (100 1 99). The extraction solvent should be completely evaporated from the layer before development of the chromatogram. This extract contains a-DNP-arginine, a-DNP-cysteic acid, a-DNP-histidine, mono-DNP-cystine, c-DNP-lysine, 0-DNP-tyrosine, di-DNP-histidine. All other DNP-amino acids are included in the ether-soluble extract. 

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