Columns fractionating

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). 

Fig. 1. Preparative separation of the components of the concentrated culture fluid on the PVT-porous glass column. (/) fraction of purified rotaviruses, (2, 3) other components of the culture fluid [32]...

Gillespie AM, Walters SM. 1986. HPLC silica column fractionation of pesticides and PCB from butterfat. J Liq Chromatogr 9 2111-2142. 

Fractions were pooled as indicated in the figure and analyzed for their sugar compositions (Table 3). The sugar compositions clearly show that most of the neutral sugars were concentrated in the large fragments that eluted close to the void volume of the column (Fraction... 

Lower panel One mg of chelator-soluble polyuronides from the indicated stages was size-fractionated on a Sepharose CL4B column, and the uronie acid content of column fractions determined. 0,7 and 20 day old fruit contain no PG, PGl only and PGl and PG2, respectively. Note the similarity of 7 and 20 day profiles despite the large amount of PG2 activity in the latter. 

On the silica gel column, fractionation of the analytes according to their polarity occurs together with the cleanup. Thus the presence of an analyte in a particular eluate gives additional information on its identity. In this respect, the silica gel column cleanup of a GPC eluate which has already been analyzed by GC with NPD or MS may be useful. The distribution of the analytes among the Eluates 1-5 is shown in the Table of the Official Method L 00.00-37. 

Figure 8.16 A simple two-dimensional gas chrtwatograph for packed column fractionation or enrichment using a Deans switch and intermediate trap for transfer to a capillary column. (Reproduced with permission from ref. 205. Copyright Dr. Alfred Huethig Publishers).

Online detection using 4H nuclear magnetic resonance (NMR) is a detection mode that has become increasingly practical. In a recent application, cell culture supernatant was monitored on-line with 1-dimensional NMR for trehalose, P-D-pyranose, P-D-furanose, succinate, acetate and uridine.33 In stopped-flow mode, column fractions can also be analyzed by 2-D NMR. Reaction products of the preparation of the neuromuscular blocking compound atracurium besylate were separated on chiral HPLC and detected by 4H NMR.34 Ten isomeric peaks were separated on a cellulose-based phase and identified by online NMR in stopped-flow mode. 

Ultraviolet spectroscopy is not as useful in detecting the -NC function. Despite its limitation, coeluting isothiocyano compounds are UV active ( 250 nm, e 1200) [27c]. Thus, a UV monitor can be interfaced with an LH-20 or silica column to detect column fractions containing -NCS compounds. Final resolution of enriched mixtures of previously fractionated isonitrile-related compounds is achieved by examining the responses generated by UV and RI detectors coupled in liquid chromatography. 

Can equipment sets be combined (e.g., replacing reactive distillation with a separate reactor and multi-column fractionation train installing internal reboilers or heat exchangers) to reduce overall system volume ... 

A clean-up step may be employed using gel permeation chromatography, Florisil, silica gel or alumina column fractionation, or solid phase extraction (SPE). 

To study the effect of the protease treatment cell-free suspension, with or without protease treatment, was subjected to gel-filtration chromatography on Sephadex G-75 and the elution patterns were compared (Fig. 1). In each case, two major peaks were detected by monitoring column fractions with absorbance at 280 nm. Degradation activities on mexacarbate, in the presence of FMN and light under anaerobic condition, were measured for each fraction. It was found that the highest activity was associated with peak II. It is interesting to note that protein (s) associated with peak II were detected with or without protease treatment these will be referred to as natural flavoprotein (B, Fig. 

Figure 9—2 shows the plant with its three reactors. The pyrolysis furnace is in the middle. At the top of the figure, the basic feeds, to the plant are shown—ethylene, chlorine, and oxygen. Ethylene and chlorine alone are sufficient to make EDC via the route on the left. The operation, call it Reaction One like Figure 9-1 does, takes place in the vapor phase in a reactor with a fixed catalyst bed of ferric (iron) chloride at only 100—125°F. A cleanup column fractionates out the small amount of by-products that get formed, leaving an EDC stream of 96—98% purity. 

The GPG spin column fractionation step is a non-equilibrium process. During the gel permeation chromatography step, the unbound small molecules in solu-... 

Analytical HPLC columns loaded with small samples gave the best separations and useful information (9). Screening the resulting column fractions with a series of enzyme substrates indicated the minimum number of different enzymes present, the salt concentrations at which enzymes tended to elute, where multiple enzymes were likely to be co-eluting (thus requiring further separation), and the substrates most useful to distinguish between the different enzymes present (Figures 2,3 and 4). 

Figure 2. Enzyme activities in analytical QM anion exchange column fractions detected with substrates selected for cellulases. Major peaks are numbered. (Reproduced with permission from ref. 9. Copyright 1990 Society of Fermentation Technology, Japan.)...

Fractions Each triacetate fraction, like the respective sample, starts with the symbol CTA (first column from left to right) and is then followed by the batch number (2nd column), fractionation number (3rd column), fraction number (Ath column) and sub-fraction number (5th column respectively). 

Unstable radionuclei result on subjecting the nuclei of some elements to neutron bombardment. During the decay process, in which the radionuclei return to more stable forms, characteristic radiation is emitted. The energy of the radiation is characteristic of the element, and its intensity forms the basis for quantitative elemental analysis. The advantages of NAA for trace analysis include low detection limits, good sensitivity, multi-element capability and relative freedom from matrix effects. However, for successful application of this technique skilled personel are required and because of the low sample throughput the amount of work involved in the analysis of column fractions, for example, is prohibitively high. In addition, it may take up to several weeks before the results are available. Further, only few laboratories have easy access to a neutron source. 

Peak Identity Confirmation by Measurement of Absorbance Ratios. It is known that peak absorbance ratio measurements provide an alternative method of peak identity confirmation to running the sample on two different columns, fraction collection for MS and so on. If Beer s law is obeyed... 

SOLVENT Choice. Solvent extraction is limited to water immiscible solvents. Solid adsorbents do not have this limitation, so miscible solvents, desirable for subsequent analytical or bioassay purposes, can be used. For example, DMSO is preferred for mutagenicity screening and has been used to elute the adsorbed organic material (211-213, 216, 235, 328). For analytical purposes, acid, base, and neutral eluents can be employed for on-column fractionation of the adsorbed organic solutes (78, 80,196). 

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