Chromatography equilibration

Determine Vq and Vt by chromatography of 0.5 ml of Soln. A. The blue fractions represent that volume which will contain the biotinylated conjugate, and the red fractions will give the waste (unbound biotin). After chromatography, equilibrate the column again with PBS. 

For the next chromatography, equilibrate the column with 10 ml of Soln. B. For storage of the column, wash with 10 ml ddH20 followed by 10 ml 20% ethanol (v/v) in ddH20. 

In principle, the experimental protocol of fluidized bed adsorption does not deviate from packed bed operations, the main difference being the direction of liquid flow. The standard sequence of frontal chromatography, equilibration, sample application, wash, elution, and cleaning (CIP) is performed with an upward direction of flow as shown in Fig. 3. During equilibration of the matrix the stabilization of the fluidized bed occurs, in case of size and/or density distribution of the adsorbent particles the classification within the bed may be detected by visual observation of the bed. As discussed below, bed stability may... 

FIG. 16-30 Operational steps in displacement chromatography, The column, initially equilibrated with a carrier solvent at time 0, is loaded with feed until time tp and supplied with displacer for a time to + tp. Development of the displacement train occurs during the time to and elution of the separated products ends at time tp. tp is the time required to remove the displacer from the column and restore the initial conditions, Components are niimhered in order of decreasing affinity for the stationary phase, [Reference Horoath et at, J, Ghromatogr, 2i8, 365 (1981). Reprinted with peimission of], Ghromatogr,]... 

Column Chromatography. The substances to be purified are usually placed on the top of the column and the solvent is run down the column. Fractions are collected and checked for compounds using TLC (UV and/or other means of visualisation). The adsorbent for chromatography can be packed dry and solvents to be used for chromatography are used to equilibrate the adsorbent by flushing the column several times until equilibration is achieved. Alternatively, the column containing the adsorbent is packed wet (slurry method) and pressure is applied at the top of the column until the column is well packed (i.e. the adsorbent is settled). 

There is no other facet where thin-layer chromatography reveals its paper-chromatographic ancestry more clearly than in the question of development chambers (Fig. 56). Scaled-down paper-chromatographic chambers are still used for development to this day. From the beginning these possessed a vapor space, to allow an equilibration of the whole system for partition-chromatographic separations. The organic mobile phase was placed in the upper trough after the internal space of the chamber and, hence, the paper had been saturated, via the vapor phase, with the hydrophilic lower phase on the base of the chamber. 

The 7a-bromo-5a-6-ketone (56) is conveniently prepared from a mixture of the 5a- and 5j5-6-ketones (55) under equilibrating conditions. It is formed from the 5a-isomer (55) via the 5a-bromo-compound, and from the 5j -isomer (55) via the 7a-bromo-5i -6-ketone (see section II-A). Dehydrobromination is effected in DMF, and chromatography of the crude product separates the title compound (58) from remaining starting material and isomeric A -6-ketone (57). ... 

The screening was performed in a way similar to that of Welch, except that it involved the use of a spectropolarimeter instead of chiral chromatography to determine the selectivity. Equal amounts of the target racemate 17 were added into each of the 16 wells containing beads and the ellipticity of the supernatant liquid in each well was measured after equilibrating for 24 h at the wavelength of the maximum adsorption (260 nm). Knowing the specific ellipticity of one enantiomerically pure... 

The first reported example31,117 involved the diethyltetraphenyl-3//-azepines 18 and 19 which were obtained in 85% overall yield by the reaction of2,3-diethyl-2//-azirine with 2,3,4,5-tetraphenylcyclopentadienone (see Section 3.1.1.1.2.). The two isomeric azepines are separable by column chromatography (alumina or silica gel), and each isomer, on warming in xylene for three days, equilibrates to a 3 8 mixture of the 3//-azepines 18 and 19. 

Step 4. Anion-exchange chromatography on a column of DEAE-Sephadex A-50. The column had been equilibrated with the basic buffer containing 0.12 M NaCl. The NaCl in the photoprotein solution was removed by gel filtration, and then the solution was added onto the column. The photoprotein adsorbed on the column was eluted with the equilibration buffer. 

A portion of the product was heated to reflux with methanolic sodium methoxide to convert it into the thermodynamic mixture of trans- (ca. 65%) and cis- (ca. 35%) isomers. Small amounts of the isomers were collected by preparative gas chromatography using an 8 mm. by 1.7 m. column containing 15% Carbowax 20M on Chromosorb W, and each isomer exhibited the expected spectral and analytical properties. The same thermodynamic mixture of isomers was prepared independently by lithium-ammonia reduction5 of 2-allyl-3-methyl-cyclohex-2-enone [2-Cyclohexen-l-one, 3-methyl-2-(2-propcnyl)-],6 followed by equilibration with methanolic sodium methoxide. 

By application of first-order, kinetic equations, B. Anderson and Degn claimed that an equilibrated (25°) aqueous solution of D-fructose contains 31.56% of jS-D-fructofuranose and 68.44% of -D-fructopyranose. N.m.r. studies, however, showed that, at equilibrium, a solution of D-fructose contains /3-D-fructopyranose, -D-fructofuranose, a-D-fructofuranose, and a trace of a-D-fructopyranose the distribution of these isomers was shown by gas-liquid chromatography to be 76,19.5, and 4%, respectively. Based on Anderson and Degn s result, Shallenberger reasoned that, as 0.68 X 1.8 = 1.22 (which approximates the reported sweetness of mutarotated D-fructose ), the furanose form(s) must possess very little sweetness. 

Molecular weight distributions were examined by high performance size-exclusion chromatography (HPSEC) as described [22] on two serial Shodex OHpak KB-803 and KB-805 columns (0.8 x 30 cm Showa Denko, Japan) with a OHpak KB-800P guard column (0.6 x 5 cm), equilibrated at 1 mL/min in 0.1 M UNO3. The system was calibrated with a pullulan calibration kit (Showa Denko). 

Ion exchange chromatographies were carried out first on a Macro-Prep High Q (IBF) column (10 ml) in Tris-HCl 20 mM (pH 8), then on a 5 ml Econo-Pac Q cartridge (BioRad) equilibrated in ammonium carbonate 20 mM buffer (pH 5) and on a 5 ml Econo-Pac S cartridge (BioRad) equilibrated in ammonium carbonate 20 rnM buffer (pH 5). 

Hydroxy-apatite chromatography was carried out on a 1.7 ml HA-Ultrogel (IBF) column equilibrated in 10 mM phosphate buffer (pH 6). Elution was performed with a linear 10-300 mM phosphate buffer (pH 6) gradient. 

FIGURE 10.5 Elution profile on OH-B12 treated by microwave heating for 6 min during silica gel 60 column chromatography. Fifty milliliters of the treated OH-B12 solution (5 mmol/1) was evaporated to dryness and dissolved in a small amount of w-butanol/2-pro-panol/water (10 7 10, v/v) as a solvent. The concentrated solution was put on a column (1.4 X 15.0 cm) of silica gel 60 equilibrated with the same solvent and eluted with the same solvent in the dark. The eluate was collected at 4.0 ml with a fraction collector. Fractions I to V were pooled, evaporated to dryness, dissolved with a small amount of distilled water, and analyzed with silica gel TLC. Inset represents the mobile pattern of the OH-B12 degradation products of fractions I to V on the TLC plate. Data are typical, taken from one of five experiments. (Reprinted with permission from Watanabe, F. et al., J. Agric. Food Chem., 46, 5177-5180, 1998. Copyright (1998) American Chemical Society.)... 

Simultaneous evolution of chromatography, as a method of analysis and separation, enables the confirmation and development of chemotaxonomic investigations of new plant species, as well as the accomplishment of quality and quantitative determinations. Thin-layer chromatography (TLC) especially proved to be very useful for analysis and isolation of small amounts of some compounds. The most significant and advantageous points of the TLC technique are its speed, cheapness, and capacity to carry out the analysis of several solutes simultaneously its continuous development under equilibrated conditions its gradient and multiple development and its ability to scale up the separation process. 

The most common approaches to sulfonylurea determinations involve high-performance liquid chromatography (HPLC). The earliest reported methods utilized normal-phase liquid chromatography (LC) with photoconductivity detection this type of detector demonstrated undesirably long equilibration times and is no longer... 

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