Extra Column Equipment

The injection system is sufficiently described by setting the appropriate boundary conditions at the entry of the pipe, for example, a rectangular pulse  

If the piping only contributes to the dead time of the plant, the delay can be described by a pipe model assuming an ideal plug flow  

When the plant behavior without the column shows non-negligible backmixing, a dispersed plug flow model might be used (Equation 6.119)  

The fluid velocity Uq inside the pipe is given by the continuity equation  

It is not necessary to model individual pipes if the cross-sections are represented by a typical diameter. In any case the primary parameter of interest is the volume (Equation 6.121) or the dead time (Equation 6.122) of the unit  

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Section 6.2 presents various models for chromatographic columns. But it has to be kept in mind that these models only account for effects occurring within the packed bed. A HPLC-plant, however, consists of several additional equipment and fittings besides the column. Therefore, the effect of this extra column equipment has to be accounted for to obtain reasonable agreement between experimental results and process simulation. Peripheral equipment (for example pipes, injection system, pumps and detectors) causes dead times and mixing. Thus, it can contribute considerably to the band broadening measured by the detector. 

Modeling Extra Column Equipment 6.3.2.1 Injection System... 

Small bore columns require equipment with very low extra-column dispersion, and this is the main difficulty associated with their use. 

Because of their small internal volume, they require equipment with low extra-column dispersion. This is not, however as serious a limitation as it is with microbore columns, and 3x3 columns can often be used satisfactorily in conventional instruments. 

It is seen from equation (34) that for a fully optimized column the maximum sample volume depends solely on the extra column dispersion (oe) This again emphasizes the importance of not only using equipment with low extra column dispersion but, also, knowing the value of (oe) for the particular chromatograph being used. 

Presently [707], we may be able to reduce the extra-column band broadening by modifying conventional H PLC equipment to a total of 1 or 2 pi. This implies that with these modifications short bulky 3 pm columns or narrow (2 mm) 5 pm columns may be used. 

Even with all these chromatographic parameters optimised, sensitivity may not be enough. The simplest way to increase sensitivity further is to inject more sample. Valve injectors are usually supplied with 20-pl loops but there are few assays for which 100-200 pi of sample cannot be injected simply by changing the loop size. A further way is to use minibore (1 mm or 2mm i.d.) or even microbore (< 1 mm) columns. Such columns give increases in sensitivity due to the narrower peaks eluted but this may reduce column loading volumes and with some equipment the extra sensitivity gained may be lost due to extra column band broadening. 

Finally, an increase in detection Knsitivity (step S) can be achieved by (1) increasing AT while holding V and constant (e.g., lower flow rates, smaller dp), (2) by decreasing k (usually by decreasing F), or (3) by reducing column diameter. With any of these approaches, the extra-column band broadening in the HPLC equipment can become important. That is, for maximum detection sensitivity, microbore -type HPLC systems may be required. 

A second problem associated with the optimized column configurations of Table XVIII is that of column length. Very short columns (L < I cm) are required for the optimal separation of larger proteins, particularly when denatured. The actual manufacture and use of very ritort columns has been solved by Walters et al. (80,7 75). However, short columns will have reduced column volumes, unless reduction in column length is compensated by increase in diameter. This in turn means smaller sample sizes must be iqjected, and the extra-column effects of the HPLC equipment must be proportionately reduced, i.e., use of microbore equipment... 

Safety devices such as air sensors and pressure transducers are built into preparative HPLC units, together with a series of valves. These devices create dead volume and contribute to the extra column volume. A large-scale chromatography unit is composed of valves for selection of buffers and feed solutions, at least two pumps, the separation column, and, in most cases, at least one detector. Instead of a fraction collector, a combination of valves is often used. These sources of dead volume create typical washout kinetics, which contribute exponentially to the band-broadening processes. For the industrial scale, the equipment is mainly customer designed. For medium scale, modular units are available [51]. Attention should be paid to extra column volume when systems are compared. Extra column effects are an important parameter of the quality of a system and should be considered when a system is purchased. 

A more serious limit to this implementation is due to the volume of the recycling pump and associated equipment such as flowmeters and pressure sensors. As the pump moves with respect to the zones, its volume leads to a dead volume dissymmetry, which can lead to a decrease extract and raffinate purities. This decrease can be significant for SMB with short columns and/or compounds with low retention. However, it can be easily overcome by using a shorter column or asynchronous shift of the inlets/outlets [54, 55]. This last solution is extremely efficient and does not induce extra costs because it is a purely software solution. 

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