Open column fractional elution

The checkers obtained roughly 30 g. of crude product in each run. Freshly opened Woelm silica gel (obtained from ICN Pharmaceuticals, 26201 Miles Ave., Cleveland, Ohio 44128) was deactivated as above, and 1800 g. was wet-packed with petroleum ether in a 65-mm. internal diameter column. In the first run the column was eluted as above, but a considerable amount of solvent was required to collect the product. Therefore, in the second run the crude product was applied to the column as a solution in petroleum ether, and 1-1. portions of 20% v/v ether petroleum ether, 30% ether, 40% ether, 50% ether, 60% ether, and 70% ether were run through. None of these six fractions contained a significant weight of material. Elution with 2 1. of 80% v/v ether petroleum ether then provided the bromohydrin acetate. 

An open column packed with neutral aluminium oxide (grade III) slurry is generally used for semi-preparative separation of large amounts of carotenoid extract, revealing three broad bands (1) carotenes and epoxy-carotenes constitute the first fraction to elute with petroleum ether, (2) monohydroxy and keto-carotenoids with 50 to 80% diethyl ether in petroleum ether are next, and (3) finally, the polyhydroxy carotenoids elute with 2 to 5% diethyl ether in ethanol or... 

Classical separations by open column chromatography with different stationary phases (silica gel, reversed-phase C-18 or C-8, polyamide, cellulose) and elution with appropriate solvent mixtures are also useful for flavonoid fractionation and purification. Different column systems can be used. The classical open column chromatography uses relatively large particle sizes (0.2-6 mm), with limited resolution, and solvent filtration through the column proceeds by the pressure of the solvent column placed on top of the stationary phase. In other cases, smaller... 

HPLC. Many HPLC methods have been described in the literature (Hornero-Mendez and Minguez-Mosquera, 1998 Schoefs, 2002, 2003, 2004 Thompson et al., 2000). When the carotenoid composition is complex, as in passiflora fruit, which contains more than 10 carotenoids, it might be necessary to first separate the different groups of carotenoids. This can be done using an open column packed with alumina. Fraction 1, which contains carotenes and epoxi-carotenoids, is eluted with petroleum ether fraction 2, which is composed of monohydroxy- and keto-carote-noids, is eluted with 70-90% diethyl ether in petroleum ether and fraction 3, made up of polyhydroxy-carotenoids, is eluted with 0—30% ethanol in ether. The pigments contained in individual fractions can be further separated using particular TLC or HPLC methods (Mercadante et al., 1998). 

Fig. 7. Fractionation by anion exchange chromatography of 1 ml portions of a 20% aqueous solution of ampicillin sodium (initial pH 8.5) kept at 22 °C for the specified periods. The DEAE-Sephadex A-25 column was eluted with 0.05 M phosphate (pH 7.4), with a linear sodium chloride gradient. Peak identities A, ampicillin B, a-aminobenzylpenicilloic acid C, dimer D, mixture of a trimer having a closed -lactam ring and a dimer having an open j -lactam ring E, tetramer /% mixture of a tetramer having an open j -lactam ring and a pen-tamer with an intact j -lactam ring G, hexamer H, octamer. (Bundgaard and Larsen 1977)...

Twenty grams of the CHa3 EtOH (3 1) fraction of the roots of H. littoralis was chromatographed on an open column with 400 g of silica gel. The column was eluted with a hexane-chloroform-methanol solvent system. 

At low concentrations, activities, a, can be approximated to concentrations. As the column is eluted with solvent, some components are removed from the stationary phase more readily than others, and separation of the mixture is achieved. Figure 4.2 shows an open-column set-up. For coloured compounds, separation can be mraiitored by eye but instrumental methods of detection (e.g. UV absorption spectroscopy or mass spectrometry) can be integrated into the system (Fig. 4.3). In column chromatography, fractions are eluted under gravity flow or under pressure (flash chromatography). 

Open-column reversed-phase chromatography has also been employed for analysis of retinol from plasma extracts, either in micro mode (50-pm diameter C18 particles, column dimensions 0.5 X 5 cm) or macro mode (column dimensions 1.5 X 6 cm), using elution with methanol water (90 10) (122). Limit of detection (fluorometric determination in collected fractions) was 0.044 pM (1.25 pg retinol/dL) for the microcolumn method, with 0.1 mL serum used. The major fluorescent interference in serum (phytofluene) was retained on the column under these elution conditions. 

Figure 2. Elution profile from columns (100 x 1.0 cm) of controlled pore glass beads of l,4-/ -linked products formed in vitro by pea membranes in 30 min. Products were dissolved in hot paraformaldehyde DMSO and eluted with DMSO in 1 ml fractions. Open circles, 1 mM UDP-[14C]glucose alone closed circles, 1 mM UDP-[14C]glucose plus 50 /iM UDP-xylose. Size markers show the molecular weight of peak elution volumes of standard dextrans, 264 = 264000 D 70 = 70000 D. (Taken with permission from Ref. 18. 1988 J. Wiley k, Sons.)...

The open-split interface dilutes eluted analytes with additional makeup gas and then splits the mixture into two fractions. An inert, fused-silica restrictor routes a portion of the sample-carrier gas mixture from the interface into the MS ionization chamber, whereas the remainder is vented to atmosphere. An open-split interface can affect the shapes and areas of peaks because solutes make contact with several items including the interface liner, the outer surfaces of the fused-silica column, and the restrictor, which can adsorb trace-level analytes if they are not properly deactivated (21). 

The need to measure concentrations in very small volumes is not restricted to biological systems. For example, open tubular columns for liquid chromatographic separations offer the advantage of increased resolution, but because their internal diameters may be as small as 15 pm, the amount of material in the eluted peaks is very small. Thus, the use of these columns requires detectors that can be used with low concentrations in small volumes. Jorgenson and co-workers showed that this could be accomplished by the insertion of a 10-pm-diameter, cylindrical electrode made from a carbon fiber into the end of the column [4]. The close fit between the column wall and the fiber ensured that a large fraction of the eluting molecules were electrolyzed. When the electrochemical data were collected in a voltammetric mode, the resolved compounds could be classi-... 

Traditionally, fractionation of the lipid extract was accomplished using open glass columns containing rather coarse and irregular silicic acid particles (9). Neutral lipids were eluted by chloroform, glycolipids were recovered using acetone, and methanol was used to obtain the phospholipids. Thus, Mounts et al. isolated PL from 5 g of oil on a 10-g column of silica gel (60-200 mesh) by sequential elution with 200 ml of chloroform, 100 ml of acetone, 100 ml of methanol, and 100 ml of 0.1% phosphoric acid in methanol (28). The last two fractions were combined to recover the phospholipid fraction. 

To elute the product RNA from the column, add 1 CV of RNA column wash buffer + 1 mM glucosamine-6-phosphate (GlcN6P) to the column and allow this to pass through, then close the column valve and allow the column to sit for 10 min at room temperature. Apply a second CV of column wash buffer + 1 mM GlcN6P, open the column valve, and collect this elution fraction. Collect two subsequent 1 CV elutions. Usually, most of the RNA comes out in the first two elution fractions (Fig. 1.4). 

Fig. 13. Left frame Manual HPLC elution (1 mL/min) of 95Nb (open circles) from a 1.7x25 mm Aminex A6 column in 0.075 M a-HiB. Pa (not shown) elutes in an identical manner. Right frame Elution of 95Zr from the same column in 0.5 M a-HiB. Only two fractions were counted (black diamonds). The dashed curve has the same shape as the elution curve in the left frame. Reproduced from [41] with the permission of Oldenbourg Verlag.

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