Dispersion in chromatographic columns

The rate theory of chromatography was introduced some 50 years ago by physicists and chemical engineers (van Deemter 1956). Despite all the work, both theoretical and experimental, that has been done since then on dispersion in chromatographic columns, the van... 

Isolation procedures for many biochemicals are based on chromatography. Practically any substance can be selected from a crude mixture and eluted at relatively high purity from a chromatographic column with the right combination of adsorbent, conditions, and eluant. For bench scale or for a small pilot plant, such chromatography has rendered alternate procedures such as electrophoresis nearly obsolete. Unfortunately, as size increases, dispersion in the column ruins resolution. To produce small amounts or up to tens of kilograms per year, chromatography is an excellent choice. When the scale-up problem is solved, these procedures should displace some of the conventional steps in the chemical process industries. 

In a chromatographic separation, the individual components of a mixture are moved apart in the column due to their different affinities for the stationary phase and, as their dispersion is contained by appropriate system design, the individual solutes can be eluted discretely and resolution is achieved. Chromatography theory has been developed over the last half century, but the two critical theories, the Plate Theory and the Rate Theory, were both well established by 1960. There have been many contributors to chromatography theory over the intervening years but, with the... 

Recalling that a separation is achieved by moving the solute bands apart in the column and, at the same time, constraining their dispersion so that they are eluted discretely, it follows that the resolution of a pair of solutes is not successfully accomplished by merely selective retention. In addition, the column must be carefully designed to minimize solute band dispersion. Selective retention will be determined by the interactive nature of the two phases, but band dispersion is determined by the physical properties of the column and the manner in which it is constructed. It is, therefore, necessary to identify those properties that influence peak width and how they are related to other properties of the chromatographic system. This aspect of chromatography theory will be discussed in detail in Part 2 of this book. At this time, the theoretical development will be limited to obtaining a measure of the peak width, so that eventually the width can then be related both theoretically and experimentally to the pertinent column parameters. 

To reiterate the definition of chromatographic resolution a separation is achieved in a chromatographic system by moving the peaks apart and by constraining the peak dispersion so that the individual peaks can be eluted discretely. Thus, even if the column succeeds in meeting this criterion, the separation can still be destroyed if the peaks are dispersed in parts of the apparatus other than the column. It follows that extra-column dispersion must be controlled and minimized to ensure that the full performance of the column is realized. 

To realistically evaluate the effect of extra-column dispersion on column performance, it is necessary to evaluate the maximum extra-column dispersion that can be tolerated by different types of columns. Such data will indicate the level to which dispersion in the detector and its associated conduits must be constrained to avoid abrogating the chromatographic resolution. 

A satisfactory chromatographic analysis demands, a priori, on an adequate separation of the constituents of the sample that will permit the accurate quantitative evaluation of each component of interest. To achieve this, an appropriate phase system must be chosen so that the individual components of the mixture will be moved apart from one another in the column. In addition, their dispersion must be constrained sufficiently to allow all the solutes of interest to be eluted discretely. At this stage it is necessary to introduce the concept of the Reduced Chromatogram. 

In a packed column the HETP depends on the particle diameter and is not related to the column radius. As a result, an expression for the optimum particle diameter is independently derived, and then the column radius determined from the extracolumn dispersion. This is not true for the open tubular column, as the HETP is determined by the column radius. It follows that a converse procedure must be employed. Firstly the optimum column radius is determined and then the maximum extra-column dispersion that the column can tolerate calculated. Thus, with open tubular columns, the chromatographic system, in particular the detector dispersion and the maximum sample volume, is dictated by the column design which, in turn, is governed by the nature of the separation. 

The chromatographic column has a dichotomy of purpose. During a separation, two processes ensue in the column, continuously, progressively and virtually independent of one another. Firstly, the individual solutes are moved apart as a result of the differing distribution coefficients of each component with respect to the stationary phase in the manner previously described. Secondly, having moved the individual components apart, the column is designed to constrain the natural dispersion of each solute band (i.e. the band... 

U.Tallarek, E. Bayer, G. Guiochon 1998, (Study of dispersion in packed chromatographic columns by pulsed field gradient nuclear magnetic resonance), J. Am. Chem. Soc. 120, 1494. 

Small bore or microbore is a term used for hplc columns that have diameters less than about 2 mm. Columns of this type were first used as long ago as 1967, but at that time the influence of extra-column dispersion was not appreciated, so that the columns were not used in chromatographs of appropriate design. In 1977 there was a renewal of interest in the properties of small bore columns, but it is only in the last few years that systems have become commercially available that allow the potential of small bore columns to be realised. Several manufacturers are now marketing a range of small bore columns, and a number of recent hplc instruments are claimed to be compatible with them. 

A value of 2.5 m for extra-column dispersion does not tell us what our system dead volume needs to be (except that it needs to be very small). Much of the extra-column dispersion in a chromatograph can be considered to occur as the solute passes through tubes, as shown in Fig. 2.3d. This effect is well understood, so that the dispersion produced can be calculated in some cases if the various dimensions are known. 

AV reciprocating tachycardia, 5 108 Axial dispersion coefficient, 10 762 Axial dispersion/mixing, 10 762-763 in adsorption columns, 2 604 in bubble tray absorbers, 2 88-89 chromatographic adsorption, 2 610 in packed column absorbers, 2 61-65 Axial dissolved oxygen profiles, 25 707-708 Axial filtration, 22 385-386 Axial-flow angular-momentum flowmeter, 22 672-673... 

As explained in Section 4.4.4, there exists an equivalency between tubular dispersion models and stagewise or tank in series models. The stagewise model, used in CHROMPLATE considers the chromatographic column to consist of a large number of well-mixed stirred tanks, arranged in series and thus represents an alternative modelling approach to that of the tubular dispersion model CHROMDIFF. The same two-component separation process is modelled and simulated in both cases. 

---