Column Properties

A number of modern HPLC columns have been used for DNA and RNA separations. One example is a silica-based C18 material available from Varian Corporation (Walnut Creek, CA). There have been recent reports on monolith polymeric materials providing excellent separations [30-32]. The remarkable performance of the DNA and RNA separation columns is based on a number of properties including the porosity of the packing material, its polarity, the absence of metal contamination, and the small size and narrow size distribution of the packing material. 

Bead pores are small relative to the nucleic acid molecule so that column interactions of the nucleic acid with the bead are on the surface of the bead. Polarity of the bead is adjusted so that nucleic acid interactions with the surface are controllable with ion pairing reagents. 

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In this chapter, the elution curve equation and the plate theory will be used to explain some specific features of a chromatogram, certain chromatographic operating procedures, and some specific column properties. Some of the subjects treated will be second-order effects and, therefore, the mathematics will be more complex and some of the physical systems more involved. Firstly, it will be necessary to express certain mathematical concepts, such as the elution curve equation, in an alternative form. For example, the Poisson equation for the elution curve will be put into the simpler Gaussian or Error function form. 

Operating temperatures are only from 10 to 30°C for TSK-GEL SW columns, but are from 10 to 80°C for TSK-GEL PW columns. The TSK-GEL SW and TSK-GEL PW sections of this chapter discuss the individual column properties and usage in more detail. 

In previous chapters, liquid chromatography column theory has been developed to explain solute retention, band dispersion, column properties and optimum column design for columns that are to be used for purely analytical purposes. The theories considered so far, have assumed that solute concentrations approach (for all practical purposes) infinite dilution, and, as a consequence, all isotherms are linear. In the specific design of the optimum preparative column for a particular preparative separation, initially, the same assumptions will be made. 

In most cases, absolute retention times are used for the identification. Modem GCs have high precision and accuracy of retention times within 0.05 %, or better can be obtained in subsequent runs during the same day. When the analysis is repeated by another instrument, the deviation may be of several percent. The reproducibility of absolute retention times is strongly dependent on the proper adjustment of all chromatographic parameters. In addition, the column properties are not exactly the same even when similar columns from the same manufacturer are used. On the other hand, absolute retention times are the most useful identification parameters if a few chemicals are to be monitored and the background is low. This method requires frequent calibration because even small changes in chromatographic conditions will influence the absolute retention times. 

Preference mapping can be accomplished with projection techniques such as multidimensional scaling and cluster analysis, but the following discussion focuses on principal components analysis (PCA) [69] because of the interpretability of the results. A PCA represents a multivariate data table, e.g., N rows ( molecules ) and K columns ( properties ), as a projection onto a low-dimensional table so that the original information is condensed into usually 2-5 dimensions. The principal components scores are calculated by forming linear combinations of the original variables (i.e., properties ). These are the coordinates of the objects ( molecules ) in the new low-dimensional model plane (or hyperplane) and reveal groups of similar... 

At variance with traditional IPRs that tend to stick very strongly to the stationary phase and to impair the initial column properties also when their presence in... 

If column properties could be considered isotropic, then we would expect symmetrical peaks of a Gaussian shape (Figure 2-1), and the variance of this peak is proportional to the diffusion coefficient (D)... 

Overall, liophilic ions (usually small ions capable for dispersive interactions) provide a useful means for selective alteration of the retention of basic analytes. Influence of these ions on the column properties is fully reversible, and equilibration requires minimal time (usually less than an hour, or about 10 to 20 column volumes). On the other hand, the mechanism of their effect is very complex and is dependent on the type of organic modifier used and on the concentration applied. Theoretical description and mathematical modeling of this process is a subject for further studies. 

To date, the shortcomings in the theoretical [22] and functional description of HPLC column properties make all these theories insufficient for practical application to HPLC method design and selection. 

Column property changes do not occur over the course of the experiment. 

Thus (m ), the mass sensitivity of the chromatographic system depends on the detector sensitivity, column dimensions, column efficiency and the capacity factor of the eluted solute. However, irrespective of the column properties, the mass sensitivity is still directly related to the detector sensitivity. It will also be seen that the column radius will depend on the extracolumn dispersion, much of which arises from the detector connecting tubes and sensor. It follows that the design of the detector and its sensitivity has a major influence on the mass sensitivity of the overall chromatographic system. 

Figure 1 Structural (left column) and dynamical (right column) properties of the systems investigated. Upper left centre-of-mass radial pair distribution function gooo( ) lower left spherical harmonic expansion coefficient g2oo(r) upper right angular velocity correlation function lower right orientational correlation function. Dotted lines CO, 80 K, 1 bar thin lines CS2, 293 K, 1 bar thick lines CS2, 293 K, 10 kbar.

Cross-linking with a bifunctional reagent. Examples of such reagents are ethyl chloroformate [7], glutaraldehyde [8] or ethyl maleic anhydride [9]. Column properties of supports prepared this way are generally low, and due to the presence of unreacted functional groups in the matrix non-specific adsorption can be high. 

Cellulose has been widely used in enzyme and antigen insolubilization although it suffers from a number of disadvantages when used for general affinity chromatography. Chiefly these are poor column properties and small pore size. One advantage of cellulose is its ready availability as a wide variety of different derivatives. 

Van Deemter 104) first related peak broadenirrg and column properties by an equation of the form... 

If the mobile phase is incompressible, as in liquid chromatography (LC), the dead volume (as so far defined) will be the simple product of the exit flow rate and the dead time. However, in LC, where the stationary phase is a porous matrix, the dead volume can be a very ambiguous column property and requires closer inspection and a tighter definition. 

Van Deemter et al. (19) related peak broadening in a gas chromatographic column to column properties through equation 9 ... 

The relevant packing properties are divided into bulk and column properties. The former pertain to the bulk powder before it is packed into the column, the latter characterize the chromatographic properties of the packed column. 

Light scattering coulter counter Particle size distribution, volume average particle diameter, number average particle diameter Stability of packed bed, hydrodynamic column properties, column performance... 

First, the column is assumed to be unidimensional. This means that it is radially homogeneous. All the column properties are constant in a given cross section and so are the concentrations of the individual components. Thus, the mass balance equations have two independent variables, the time t and the column length z. We shall consider two functions of these variables, the concentrations — C, in the mobile phase and Cg, in the stationary phase. 

For the actual user, the desire is to obtain well-resolved peaks in a short time period. How this might be done requires an understanding of how column properties, analyte equilibrium properties, and resolution are interrelated. To do this, first we will introduce a new term, a or the selectivity factor, which represents the equilibrium distribution between two analytes, A and B. 

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