Chromatography microscope slide

Figure 9.29 Membrane formation by meteoritic amphiphilic compounds (courtesy of David Deamer). A sample of the Murchison meteorite was extracted with the chloroform-methanol-water solvent described by Deamer and Pashley, 1989. Amphiphilic compounds were isolated chromatographically on thin-layer chromatography plates (fraction 1), and a small aliquot ( 1 p,g) was dried on a glass microscope slide. Alkaline carbonate buffer (15 p,l, 10 mM, pH 9.0) was added to the dried sample, followed by a cover slip, and the interaction of the aqueous phase with the sample was followed by phase-contrast and fluorescence microscopy, (a) The sample-buffer interface was 1 min. The aqueous phase penetrated the viscous sample, causing spherical structures to appear at the interface and fall away into the medium, (b) After 30 min, large numbers of vesicular structures are produced as the buffer further penetrates the sample, (c) The vesicular nature of the structures in (b) is clearly demonstrated by fluorescence microscopy. Original magnification in (a) is x 160 in (b) and (c) x 400.

Use plastic sheets precoated with silica gel for thin-layer chromatography. One piece the size of a microscope slide should suffice. Spot crude... 

The crude carotenoid is to be chromatographed on a 12-cm column of acid-washed alumina (Merck), prepared with petroleum ether (37-53°C) as solvent. Run out excess solvent, or remove it from the top of the chromatography column with a suction tube, dissolve the crude carotenoid in afew milliliters of toluene, and then transfer the solution onto the chromatographic column with a Pasteur pipette. Elute the column with petroleum ether, discard the initial colorless eluate, and collect all yellow or orange eluates together in a 50-mL Erlenmeyer flask. Place a drop of solution on a microscope slide and evaporate the rest to dryness (rotary evaporator or aspirator tube). Examination of the material spotted on the slide may reveal crystallinity. Then put a drop of concentrated sulfuric acid beside the spot and mix with a stirring rod. Compare the color of your test with that of a test on the other carotenoid. 

Fig. 3. Thin-layer chromatography in superfine Sephadex G-200 on microscope slides, followed by diffusion against monospecific antisera to identify proteins as indicated.

Experimental details solid-state photolysis 957 A crushed crystalline ketone (279a or 279b) ( 5 mg), suspended in hexane (3 ml), was placed between Pyrex microscope slides, sealed in a polyethylene bag under nitrogen and irradiated with a medium-pressure mercury lamp (450 W) at a distance of 10 cm from a water-cooled Pyrex immersion well (Figure 3.9) at either 20 or — 20 °C (cryostat ethanol bath). The product, a chiral organic salt, was derivatized to the corresponding methyl ester by treatment with excess diazomethane and purified by column chromatography. 

Like paper chromatography, thin-layer chromatography is a form of plane chromatography in that the stationary phase is held on a plane rather than in a column. Table 12.1 lists important stationary phases used in TLC along with the respective predominant sorption process operative with each of them. The solid phase is supported on to glass, metal or a plastic substance. (Microscope slides... 

At the end of the 1930s, adsorption chromatography in columns as introduced by Tswett had become a powerful separation technique for plant extracts and natural products. Simultaneously, the need for a more rapid alternative suitable for identification of separated substances led to the invention of an open chromatographic system. In 1938, Izmailov and Shraiber reported the separation of belladonna alkaloids on a thin adsorbent layer, coated onto microscopic slides. Development of circular chromatograms was achieved by placing small amounts of various solvents to the center of samples previously applied as spots onto the layer. This method was an extremely rapid microtechnique requiring only small amounts of stationary and mobile phases. 

Thin-layer chromatography can establish that two compounds suspected to be identical are in fact identical. Simply spot both compounds side by side on a single plate and develop the plate. If bofh compounds fravel fhe same distance on the plate (have the same Rj value), they are probably identical. If fhe spot positions are not the same, the compounds are definitely not identical. It is important to spot compounds on the same plate. This is especially important with hand-dipped microscope slides. Because they vary widely from plafe to plate, no two plates have exactly the same thickness of adsorbent. If you use commercial plates, this precaution is not necessary, although it is nevertheless a good idea. 

The solid stationary phase is usually alumina (Al O ) or silica (Si02), which is made into a slurry with water and spread onto a microscope slide. This is then put into an oven, where it dries out into a solid white coating on the glass. A chromatogram is then made in a similar way to paper chromatography (Figure 29.7). 

---