in black and white
Main menu
Share a book About us Home
Biology Business Chemistry Computers Culture Economics Fiction Games Guide History Management Mathematical Medicine Mental Fitnes Physics Psychology Scince Sport Technics

Chromatographic scince series - Cazes J.

Cazes J. Chromatographic scince series - Marcel Dekker, 1996. - 1098 p.
ISBN 0-8247-9454-0
Download (direct link): сhromatography1996.pdf
Previous << 1 .. 164 165 166 167 168 169 < 170 > 171 172 173 174 175 176 .. 601 >> Next

If the compounds themselves are not visible or fluorescent, detection can be performed by use of iodine vapor in a closed chamber; this technique can be used for visualization of substances from a large variety of chemical classes as dark or light brownish zones on a tan background. In most instances the iodine can be evaporated after the compound spots have been marked, leaving the desired compounds chemically unchanged.
If destructive reagents (e.g., vanillin-sulfuric acid) are necessary for detection of the separated compounds, a vertical channel must be scraped in the layer about half a centimeter from the edge of the streak. After covering the major portion of the layer with a suitable glass plate, the part of the
layer which is not covered is sprayed, and thus serves as a guide area. If heating is necessary for detection, the sprayed portion of the plate must be detached from the rest, by use of a glass cutter, since heating the developed preparative plates can lead to decomposition of the compounds of interest.
After location of the desired compound, the subsequent steps are mechanical removal of the adsorbent zone, extraction of the compound from the stationary phase with a suitable solvent, separation from the residual adsorbent, and concentration of the solvent. The areas of the layer containing the compounds of interest are scraped off cleanly down to the glass with a suitable scraper or spatula.
Several commercially available inexpensive devices and individually developed methods exist for extracting the compounds from the stationary phase; these are summarized in (14-16). Vacuum collectors are particularly recommended. This method is not very practicable for sensitive substances because the stationary phase containing the desired compounds is in constant contact with a stream of air, and there is some risk of oxidation. In our experience one of the best methods is to put the adsorbent with the compound to be extracted in an empty receptacle containing a sintered glass filter to retain the adsorbent and to extract the compound with a suitable solvent with the help of vacuum.
The substance should be highly soluble in the solvent or solvent mixtures used to extract a compound; the solvent should also be as strong as possible (polar for silica gel), but free from water and methanol. If water is the chosen solvent, it should be removed by lyophilization. Since silica is significantly soluble in methanol, and some of its common impurities are also soluble, this solvent should be avoided. Chloroform is widely used for apolar substances, and ethanol or acetone for polar compounds. The mobile phase used for the separation is highly recommended for extraction also. As a rule of thumb the volume of solvent (11) required when the chromatographic mobile phase is chosen for extraction is (43):
Vsolvent = 10 X (1.0 - Rf) X Vscraped
It should be noted that the longer the substance is in contact with the adsorbent, the more likely is decomposition to occur. Once the solution of the compound to be isolated is obtained (free from adsorbent), the extract must to be evaporated to dryness. The evaporation temperature should be as low as possible, to avoid chemical decomposition.
C. Special Techniques
1. Gradient of Layer Thickness
A common problem in CPLC is the loss of resolution compared with analytical TLC. The two reasons for this are the wide range of particle size distribution of the stationary phase, and the layer thickness. Use of the Uniplate-T taper plate (44) provides improved resolution; spot elongation and overlapping are'greatly reduced as a result of the gradient effect of layer thickness. This plate has a wedge-shaped layer; it is thin (0.3 mm) at the bottom and thick (1.7 mm) at the top. The preadsorbent part of the layer has a thickness of 0.7 mm. A schematic drawing of this plate is shown in Figure 6. The improved performance of the taper plate is similar to the improved resolution in the lower Rf range which results from the use of circular TLC.
The cross-sectional area traversed by the mobile phase front increases during development. The cross sectional flow per unit stationary phase area is, therefore, always highest at the bottom of the layer, decreasing toward the mobile phase front. As a result, the lower portion of a spot moves faster than the top portion, keeping each component focused in a narrow band. Band broadening is significantly reduced, especially for compounds with higher Rf values. Compounds with lower Rf values are subject to greater mobile phase velocity relative to higher Rf compounds than on conventional plates. This is because of the increase in solvent front size with migration distance.
Preadsorbent Barrier Adsorbent
Figure 6 Schematic diagram of the Uniplate-T taper plate.
Because of this, the distance between bands at lower Rf values is increased, providing better separations.
Previous << 1 .. 164 165 166 167 168 169 < 170 > 171 172 173 174 175 176 .. 601 >> Next