Column overload conditions

The co-injection of a racemate (e.p. d,/-cxo-norborneol) and a volatile resolving agent e.g. d-camphor) into a non-optically active g.l.c. column can lead to the resolution of the enantiomeric pair. The choice of resolving agent rests with its ability to interact with the enantiomeric pair (providing diastereoisomeric combinations)— particularly in the column-overload condition at the early part of the elution— and yet be chromatographically resolved from them. 

Contemporary development of chromatography theory has tended to concentrate on dispersion in electro-chromatography and the treatment of column overload in preparative columns. Under overload conditions, the adsorption isotherm of the solute with respect to the stationary phase can be grossly nonlinear. One of the prime contributors in this research has been Guiochon and his co-workers, [27-30]. The form of the isotherm must be experimentally determined and, from the equilibrium data, and by the use of appropriate computer programs, it has been shown possible to calculate the theoretical profile of an overloaded peak. 

Column dimensions mainly determine the quantity of sample to be separated. However, because the SEC process is driven by size separation and is diffusion controlled, special care has to be taken to keep optimized separation conditions, especially when going to smaller internal diameter columns. Overloading and excessive linear flow rates can be observed quite often in these typese of columns. For this reason, standard 8-mm i.d. columns are commonly used, as they are rugged and have a good tolerance toward separation conditions. 

A better solution for preparative columns is the development of separation media with substantially increased selectivities. This approach allows the use of shorter columns with smaller number of theoretical plates. Ultimately, it may even lead to a batch process in which one enantiomer is adsorbed selectively by the sorbent while the other remains in the solution and can be removed by filtration (single plate separation). Higher selectivities also allow overloading of the column. Therefore, much larger quantities of racemic mixtures can be separated in a single run, thus increasing the throughput of the separation unit. Operation under these overload conditions would not be possible on low selectivity columns without total loss of resolution. 

To determine the band dispersion that results from a significant, but moderate, sample volume overload the summation of variances can be used. However, when the sample volume becomes excessive, the band dispersion that results becomes equivalent to the sample volume itself. In figure 10, two solutes are depicted that are eluted from a column under conditions of no overload. If the dispersion from the excessive sample volume just allows the peaks to touch at the base, the peak separation in milliliters of mobile phase passed through the column will be equivalent to the sample volume (Vi) plus half the base width of both peaks. It is assumed in figure 10 that the efficiency of each peak is the same and in most cases this will be true. If there is some significant difference, an average value of the efficiencies of the two peaks can be taken. 

Unfortunately, this procedure can only be successful if the critical pair can be well resolved and column overload is a practical solution to the problem. Often, due to the complex nature of practical mixtures, values for these conditions are not realized and the optimum column... 

Figure 4.40 Separation of alkylbenzyl alcohol Isoeers by reversed-phase senipreparative HPLC In a nass overload condition. The dotted line is the equivalent analytical sepeuration on the same column. (Copyright Whatman Inc.)...

Xt is very difficult to provide an optimum set of conditions for operation of a liquid chromatograph under overload conditions due to the coiq>lex interactions among a large number of parameters [591,592,595,608,620,625]. The following general observations seem to be applicable in most cases. The column efficiency should be as high as possible and separatimis should be carried out using concentration overload conditions. The production rate of a... 

When translated to the SMB conditions, these features imply that increasing feed concentration lead to an increasing degree of non-linearity due to the fact that the adsorption columns increasingly are operated under overload conditions. This effect is predicted by the approach summarized in the previous section, in particular by Eqs. (8) to (19), which allow the calculation of the constraints on m1 and m4 and the boundaries of the complete separation region in the (m2, m3) plane as a function of feed composition [19]. 

Linearity of response versus absolute amount injected must be confirmed for each different sample type and for each different set of chromatographic operating conditions. This linearity cannot be assumed. Nonlinearity may result from column overload, detector overload, or adsorption problems. 

A generic gradient recommended for preparative separations is to start the elution at a concentration of B that corresponds to a value 10% earlier that the anticipated desorption then apply a change in concentration of B corresponding to 5% over 15 minutes. To exemplify this approach Table 5.2 shows a gradient, applied to an analytical column, used to purify a peptide that is anticipated to desorb at 20% B under overload conditions. 

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