Preparative chromatography collection

Preparative chromatography involves the collection of individual solutes as they are eluted from the column for further use, but does not necessarily entail the separation of large samples. Special columns can be designed and fabricated for preparative use, but for small samples the analytical column can often be overloaded for preparative purposes. Columns can be either volume overloaded or mass overloaded. Volume overload causes the peak to broaden, but the retention time of the front of the peak... 

The dimensions of the exit tube from the detector are not critical for analytical separations but they can be for preparative chromatography if fractions are to be collected for subsequent tests or examination. The dispersion that occurs in the detector exit tube is more difficult to measure. Another sample valve can be connected to the detector exit and the mobile phase passed backwards through the detecting system. The same experiment is performed, the same measurements made and the same calculations carried out. The dispersion that occurs in the exit tube is normally considerably greater than that between the column and the detector. However, providing the dispersion is known, the preparative separation can be adjusted to accommodate the exit tube dispersion and allow an accurate collection of each solute band. 

When the reaction has progressed to the desired stage (Note 6), the flow of air is stopped and the mixture is filtered. After the filtrate has been extracted with two 350-ml. portions of petroleum ether (b.p. 30-60°), the combined hydrocarbon extracts are washed successively with two 100-ml. portions of 2N hydrochloric acid and three 100-ml. portions of water. The petroleum ether is distilled from the solution, heated in a water bath, through a 60-cm. Vigreux column, and the residual liquid is distilled under reduced pressure. The fraction, b.p. 64-65° (1.0 mm.) or 132-134° (35 mm.), is collected as 39.5-52.0 g. (64-85%) of colorless liquid, 1.4846-1.4850. This distillation fraction contains (Note 6) 80-90% of the ci5-cyclododecene (51-76%) accompanied by 10-20% of a mixture of cyclododecane and ai,ira s-l,5-cyclododecadiene (Note 7). If desired, the cis-cyclododecene may be further purified by preparative chromatography or separation of the silver nitrate-olefin addition complex (Note 8). 

Figure 5. Composite MWD diagram showing the calculated distributions of four of the five master fractions obtained from preparative chromatography of organosolv aspen lignin (fraction number 4 to 1 from left to right). The Mw values for the master fractions from left to right are 1,110, 1,310, 2,420, and 8,050, respectively. The insert shows the elution profile of organosolv aspen lignin from the YMC preparative /z-Styragel column (5 x 200 cm). The 30 fractions collected were pooled into the five master fractions shown.

The instrument needed to perform automated preparative chromatography (which will hereafter be referred to as autoprep) in the desired format is based upon a standard high-pressure binary gradient instrument. The most important feature of the instrument for our particular requirements was the ability to operate from the microtitre plate sample format. This is important for combinatorially derived samples as it appears to be the standard format. It also becomes the most sensible arrangement for collecting large numbers of fractions within a reasonable size collection tray footprint . 

The main fraction collected from a sample injection of 100 mg was returned to the synthesising chemist for recovery from the solvent. Only 40 mg of pure compound was recovered and this was considered unacceptable by the chemist (especially since the analytical trace shown in Fig. 8..3 suggested a purity, from a normalised UV chromatogram, somewhere in the region of 80 — see discussion later). Seemingly poor practical sample recoveries (compared to expected recoveries) are not uncommon in preparative chromatography and systematic examination of the process is required to determine the reason for this as and when it occurs. 

The analysis of the mobile-phase fractions collected at the outlet of column is the oldest method used in CCC (droplet countercurrent chromatography and rotation locular countercurrent chromatography) to elucidate the quality of separation and to characterize solutes. With modern CCC, such as CPC, CCC type J, and crossaxis, numerous apphcations have been described for preconcentration and preparative chromatography. 

Automation allows batch chromatography to be run as a continuous process. Multiple injections using a separate pump and fraction collection provide an opportunity for continuous unattended operation. In iso-cratic separations, sample injection is often made before previously injected product elutes from the column, thus reducing cycle time and solvent consumption. Continuous and automated processes are always used with smaller columns and lower amounts of expensive enantioselective stationary phases. One of the future goals for modern PHPLC optimization would be the creation of software that would allow computer simulation modeling of nonlinear effects in preparative chromatography. 

There are two main areas in which preparative chromatography is used [13], The first area is to obtain purified components in order to collect data and analyze them further the second area is the production of the final material [13], In the first area, the purified chemicals are needed as intermediates in the process generating the desired information. This is the case when relatively small amounts of material are needed for identification and characterization, like the case in paper II, or for the acquisition of toxicological or pharmacological data. Time is important in such cases and very little time is available for optimization [13], In such cases the time spent by the person developing and executing the separation is the essential component of cost, and that time should be minimized. The contribution to the cost from solvents, stationary phases, chemicals and instruments will be small in comparison, due to the small amounts required. In such cases, there is no time available nor much purpose for isotherm measurements and modeling [13], In the second area, a purified compound is needed to obtain a final product,... 

Because of the different nature of the separation problems encountered in chemistry, a variety of names are used, sometimes interchangeably, e.g., separation, concentration, enrichment, extraction, purification. Anal5deal with dilute solutions, and separation is usually the only term used, complemented by resolution when the degree of separation achieved is quantified. In preparative chromatography, extraction usually refers to the collection, from the feedstock, of a certain component contained in that feed, or of most of it. Usually, the extraction is positive that is, the extracted component is desirable. However, in some cases, an xmdesirable component can be eliminated from a mixture the procedure is quite similar, and chromatography is equally well suited to extraction and to elimination. 

Cut points Times, mobile phase volumes, or threshold values of the concentration when fraction collection begins or ends in preparative chromatography. 

Rotating annular column An implementation of preparative chromatography using a cylindrical, annular column rotating aroimd its axis. A continuous feed stream is injected at a fixed position just above the packing. Each component band follows a helical trajectory and is collected as a continuous stream along a fixed sector of the outlet annulus. 

Simulated Moving Bed An implementation of preparative chromatography in which a series of identical columns is used. The continuous injection of a feed stream and of the mobile phase stream, the continuous collection of two fraction streams are made in positions which are periodically moved by one column length. The result is a semisteady concentration profile for each component, which oscillate slowly, and permits the collection of two streams of constant composition. The process is equivalent to moving the stationary phase down the column while the mobile phase flows upward, the faster moving component eluting at the top, with the mobile phase, the slower moving one at the bottom, writh the stationary phase. 

After the mobile phase exits the column, possibly carrying analytes, it will almost always go through one or more detectors (the only exception likely would be that the mobile phase might be collected in a fraction collector as might be done in preparative chromatography). Detection is the topic of the next section. 

In preparative liquid chromatography the technique is dependent on the amount of substance to be processed. In biochemical analysis preparative chromatography is most often used in order to collect, for spectroscopic identification, a sample of unknown substances. This can be done with quite small amounts of substance, and thus repeated sample collection from analytical columns is sufficient. If preparative... 

Chromatography is primarily a separation technique. If it is applied for the collection of pure material then it is called preparative chromatography, and if one fractionates with the aid of a pump on fine-particle columns, it is called modern preparative liquid chromatography. The aim is to extract as much material in as pure a state as possible. 

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