Prefractionation

Prefractionation by liquid-solid chromatography may be performed by HPLC, flash chromatography, or solid phase extraction (SPE). In order to achieve sufficient resolution to produce high purity screening libraries (1-5 compounds per well), a combination of two opposing or orthogonal steps is typically required. 

The preparation of prefractionated natural product libraries for drug discovery can be made more efficient by automation. Automation reduces manpower requirements, improves efficiency and increases productivity. In addition, human errors are reduced and reproducibility is improved. Overall, automation increases throughput and can produce larger and more diverse screening libraries. 

Despite the excellent resolving powers of modem capillary chromatography, there are situations where the analyst may chose to prefractionate the aroma isolate. Some of the more common methods to prefractionate flavor isolates prior to GC analysis include acid/base separations, high pressure liquid chromatography (HPLC), silicic acid column chromatography and preparative GC. 

Acid/base/neutral fractionation of aqueous or organic solvent-based aroma isolates are relatively simple to accomplish taking advantage of the effect of pH on the solubility of ionizable analytes. One can, by changing pH, selectively partition an aroma isolate into acid, basic, and neutral fractions. 

An HPLC method is attractive for flavor fractionation since it uses a different set of physical properties for separation than GC does. A flavor isolate may be separated by adsorption or reverse/normal phase chromatography. Adsorption chromatography is a good initial choice since it has the greatest column capacity and can handle the widest range of types of compounds [53]. Fractions based on adsorption affinity could then be further fractionated on a normal or reverse phase column [54]. 

A simple inexpensive method for flavor fractionation is via silicic acid [55]. Basically, a flavor concentrate is passed through a column of sihcic acid and then eluted with a solvent gradient. This effectively fractionates the flavor isolate by compound polarity. The major problem with this method is potential artifact formation. However, careful control of the activity of the silicic add and limiting contact time minimizes artifact formation. 

Preparative GC offers the greatest resolving power of the fractionation methods. Traditionally, a flavor concentrate was chromatographed on a 0.125-inch or 0.25-inch o.d. packed column and the effluent collected via cold trap in numerous fractions. The solvent-free fractions could be subjected to sensory evaluation at this point to focus on a specific sensory property (e.g., an off-flavor or desirable note) or subjected to additional chromatographic separations. Today it is more common to use large 

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Consider a three-product separation as in Fig. 5.11a in which the lightest and heaviest components are chosen to be the key separation in the first column. Two further columns are required to produce pure products (see Fig. 5.11a). However, note from Fig. 5.11a that the bottoms and overheads of the second and third columns are both pure B. Hence the second and third columns could simply be connected and product B taken as a sidestream (see Fig. 5.116). The arrangement in Fig. 5.116 is known as a prefractionator arrangement. Note that the first column in Fig. 5.116, the prefractionator, has a partial condenser to reduce the overall energy consumption. Comparing the prefractionator arrangement in Fig. 5.116 with the conventional... 

Figure 5.11 Choosing nonadjacent keys leads to the prefractionator arrangement.

Figure 5.13 Compasition profiles for the middle product in the prefractionator arrangement show that there are no remixing effects. (From Triantafyllou and Smith, Trans. IChemE, part A, 70 118, 1992 repr uced by permission of the Institution of Chemical Engineers.)...

In addition, one other feature of the prefractionator arrangement is important in reducing mixing effects. Losses occur in distillation operations due to mismatches between the composition of the column feed and the composition on the feed tray. Because the prefractionator distributes component B top and bottom, this allows greater freedom to match the feed composition with one of the trays in the column to reduce mixing losses at the feed tray. 

The elimination of mixing losses in a prefractionator arrangement means that it is inherently more efficient than an arrangement using simple columns. 

Consider now thermal coupling of the prefractionator arrangement from Fig. 5.116. Figure 5.16a shows a prefi-actionator arrangement with partial condenser and reboiler on the prefractionator. Figure 5.166 shows the equivalent thermally coupled prefractionator arrangement sometimes known as a Petlyuk column. To make the two arrangements in Fig. 5.16 equivalent, the thermally coupled prefractionator requires extra plates to substitute for the prefractionator condenser and reboiler. 

Figure 5.17 The thermally coupled prefractionator can he arranged in a single shell.

Introduce complex distillation configurations. Introduce prefractionation arrangements (with or without thermal coupling), side-rectifiers, and side-strippers to the extent that operability can be... 

Prefractionator arrangements (both with and without thermal coupling) can be used to replace either direct or indirect pairings. 

In this case, a preliminary separation will have taken place either in the plant by stabilization, or by the chromatograph which will have had a prefractionating column. This column will isolate the components having boiling points higher than pentane, allowing only the noncondensable hydrocarbons and a fraction of the pentanes to pass through to the analytical column. 

Eig. 4. Mobil—Badger process for ethylbenzene production H = heater Rx = reactor P = prefractionator BC = benzene recovery column ... 

Extractive distillation, using similar solvents to those used in extraction, may be employed to recover aromatics from reformates which have been prefractionated to a narrow boiling range. Extractive distillation is also used to recover a mixed ben2ene—toluene stream from which high quaUty benzene can be produced by postfractionation in this case, the toluene product is less pure, but is stiU acceptable as a feedstock for dealkylation or gasoline blending. Extractive distillation processes for aromatics recovery include those Hsted in Table 4. 

Following the backflush of the primary column and separation of the analytes on the second column, the system can then be returned to its original prefractionation position, ready for the next sample injection. 

Most of the benzene in the gasoline pool comes from the reformer unit (reformate). To reduce the reformate s benzene, one must modify the feedstock quality and/or operating conditions. Benzene s precursors in the reformer feed (C, and C ) can be prefractionated and sent to an isomerization unit. The reformer operating pressure can be reduced... 

For the same reason it is also possible to use Over Pressure Layer Chromatography (OPLC) on-line for prefractionation or as a clean-up method for HPLC [13,14] A group separation according to polarity is followed by a differentiation of the substances according to their differing lipophilicities (Fig. 5). 

Table 4.45 shows the main features of SEC. This technique has become an indispensable tool for polymer characterisation. SEC has some advantages over other LC methods, such as the predictability of the end of a chromatographic run and of the retention times in a calibrated chromatographic system. SEC is an attractive technique for prefractionation or sample clean-up prior to a more sensitive RPLC technique. This intermediate step is especially interesting for experimental purposes whenever polymer matrix interference cannot be separated from the peak of interest [647]. Disadvantages are that the whole separation must be eluted within the... 

In on-line multidimensional HPLC (MDHPLC) two relatively high-efficiency columns are coupled in an instrument, via the use of valves, traps and other means. In LC-LC the precolumn is used for sample cleanup and prefractionation, before introduction of the fraction of interest to the analytical column. Much of the instrumentation for MDHPLC is the same as that in conventional one-dimensional experiments. However, the additional complexity of MDHPLC experiments leads to greater difficulties than those found in conventional HPLC ... 

SEC in combination with multidimensional liquid chromatography (LC-LC) may be used to carry out polymer/additive analysis. In this approach, the sample is dissolved before injection into the SEC system for prefractionation of the polymer fractions. High-MW components are separated from the additives. The additive fraction is collected, concentrated by evaporation, and injected to a multidimensional RPLC system consisting of two columns of different selectivity. The first column is used for sample prefractionation and cleanup, after which the additive fraction is transferred to the analytical column for the final separation. The total method (SEC, LC-LC) has been used for the analysis of the main phenolic compounds in complex pyrolysis oils with minimal sample preparation [974]. The identification is reliable because three analytical steps (SEC, RPLC and RPLC) with different selectivities are employed. The complexity of pyrolysis oils makes their analysis a demanding task, and careful sample preparation is typically required. 

Overpressure layer chromatography (OPLC) can be employed as a cleanup method for HPLC, or used for prefractionation. OPLC-HPLC coupling has been described by ref. [1003],... 

Because of the difficulties in abundance and compatibility described above, fractionation steps are often performed on protein mixtures prior to 2D gel separation to reduce the complexity of the mixtures. Prefractionation of proteins can be achieved by (i) isolation of cell compartments such as the plasma membrane or organelles such as mitochondria or nuclei, (ii) by... 

A number of affinity-based or chromatography methods have been used to prefractionate protein samples for 2D electrophoresis. For example, proteins of low abundance can be enriched from crude lysates by affinity-... 

This remixing that occurs in both sequences of simple distillation columns is a source of inefficiency in the separation. By contrast, consider the prefractionator arrangement shown in Figure 11.9. In the prefractionator, a crude split is performed so that Component B is distributed between the top and bottom of the column. The upper section of the prefractionator separates AB from C, whilst the lower section separates BC from A. Thus, both sections remove only one component from the product of that column section and this is also true for all four sections of the main column. In this way, the remixing effects that are a feature of both simple column sequences are avoided4. 

The elimination of mixing losses in the prefractionator arrangement means that it is inherently more efficient than an arrangement using simple columns. The same basic arguments apply to both distributed distillation and prefractionator arrangements, with the additional degree of... 

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