Extra Column Effects

The important parameters to consider are the selectivity (dKJdlogR), the ratio of pore volume, Vp, over void volume, Vq, the plate height, H, and the column length, L. The distribution coefficient, Kq, has a slight effect on resolution (with an optimum at Kp 0.3-0.5). In addition to this, extra column effects, such as sample volume, may also contribute to the resolution. 

Kirkland, J. J., Yau, W. W., Stoklosa, H. J., and Dilks Jr., C. H., Sampling and extra-column effects in high-performance liquid chromatography influence of peak skew on plate count calculations, /. Chromatogr. Sci., 15, 303, 1977. 

The extra-column dispersion governs the dimensions of the column that we use. In the calculation above, the dispersion is increased by about 8% by the extra-column effects. If we want the dispersion to be increased by no more than this, then should not be any smaller than the value calculated above. This in turn limits the retention volume, and thus the volume of the column itself. The minimum column volume we can use will depend on the amount of extracolumn dispersion and on what we consider to be an acceptable increase in peak width that is produced by extra-column effects. In practice, this acceptable increase is taken as 10%, based on an unretained solute, and if we take 50 (i as a typical figure for extracolumn dispersion then the minimum column diameter works out at about 4.5 mm for a column 25 cm long. 

It has been shown that when the intracolumn effect of mass transfer and diffusion is the main factor controlling band broadening, the column efficiency decreases with the increase of the viscosity of the meth-anol/water mixture on the other hand, when the extra-column effect is the main factor, an increase in viscosity of the eluents will help in improving column efficiency. Column efficiency is also related to the properties of the sample [86]. 

However, those who have worked with smaller diameter columns have often experienced lower performance and other difficulties. This is primarily due to extra-column effects the bandspreading in the injector, detector and tubing, or the gradient delay volume of the instrument. Troubleshooting guidelines for sorting out the causes of these difficulties are available in Reference f. With proper care, 2-mm columns can be run on a standard modern HPLC instrument with few difficulties. Smaller i.d. columns require special instrumentation. 

To fully utilize small particles (i.e., less than 2 pm) packed in columns greater than 50 mm in length, an HPLC system must be developed that can operate at high pressures. For the best results using columns with 1-2-pm particles, extra-column effects, dwell volumes, and detectors must be optimized for optimum performance. Additionally, commercial... 

A transfer function, defined as the Laplace transfer of the impulse response of a linear system, can be obtained from the model. This can be very useful, because with a transfer function the influence of extra-column effects (detector, amplifier, filter) on the peak shape can be easily calculated. The transfer function is ... 

The stochastic model applies to processes involving the stationary phase. To analyze the chromatogram, we need to subtract contributions to peak broadening from dispersion in the mobile phase and extra-column effects such as finite injection width and finite detector volume. These effects account for the width of the unretained peak. To subtract the unwanted effects, we write... 

A rule of thumb is that the injection volume can be as high as 30% of the volume of a peak that elutes from the column when using a small injection (e.g., 10 juL) and there should be no significant broadening of the peak with this larger injection. To understand this rule of thumb, one must consider the contributions to band broadening. At injection, the sample volume will be diluted to a volume that is mainly dependent upon the efficiency of the column. The final peak width, Wpeak, observed in the detector is a result of the volume of the sample injected and of the spreading from the column, the detector, and the extra column effects. 

In capillary gel electrophoresis, one of the major contributors to band broadening, besides the injection and detection extra-column effects, is the longitudinal diffusion of the solute molecules in the capillary tube [14], The theoretical plate number (N) is characteristic of column efficiency ... 

The use of nonporous supports for protein immobilization will eliminate the problems associated with the intraparticle diffusion limitations. With columns regularly packed with small spherical particles of limited size distribution, the mass transfer effects associated with a nonuniform flow distribution will be minimized. Extra column effects due to diffusion in the stagnant liquid pockets will be reduced by using short and properly designed connecting lines to the injector and the detector. 

Eigure 17-17. Theoretical plates versus retention factor for series of parabens separated by isocratic chromatography. Extra-column effects result in decreased efficiency for early-eluting components (low values of k). 

The extra-column effects (due to sampling loop, connecting elements, detector, etc.) were determined experimentally and taken into account by the convolution integral during the time domain fitting of experimental column responses. 

The developed method takes into account extra column effects of the measuring system. It was also confirmed that Peclet numbers for equally... 

As previously indicated, this discussion is organized for chromatograms from very narrow polymer standards for which we can consider that the effect of molecular weight distribution is negligible and for which the unique separation process is size exclusion. With these limitations, the contribution to band broadening is conveniently separated into extra column effects, eddy dispersion, static dispersion, and mass transfer. In the most classical chromatographic interpretation, extra-column effects are not discussed and the three other contributions are considered as Gaussian, so there is simply the addition of their variances. The number of theoretical plates is defined as N = VJaY and the influence of v, the linear velocity of the eluent, is summarized by the so-called Van Deemter equation ... 

Fig. 1 Extra-column effects on the chromatogram, i) Normal chromatogram for a test separation ii) chromatogram obtained by inserting 75 pL of extra volume between the column and the detector inlet tube iii) chromatogram obtained by inserting 2 mL of extra volume between the eolumn and the detector inlet tube.

So-called plant dispersion" or extra column effects" have to be taken into account by additional mathematical models rather than including them indirectly in the model parameters of the column, e.g. by altering the dispersion coefficient. The combination of peripheral and column models is easily implemented in a modular simulation approach. In a flowsheeting approach the boundary conditions of different models are connected by streams (node balances) and all material balances are solved simultaneously. 

Determine extra column effects such as dead volume and the backmixing of the plant... 

Use preparative materials for the adsorbent and, if possible, semi-preparative columns. Always check consistency of the results with those of the initial tests (pressure drop, fe ). Use one plant for all measurements and try to keep extra column effects low. 

Use one plant for all measurements and try to keep extra column effects low. Determine all dead volumes of plant and column. 

Peak deformation through extra column effects of the plant cannot be accounted for... 

Band broadening can occur in other parts of the chromatographic system as well as in the column. Contributions to this extra-column broadening may come from the injector, the detector flow cell, and the connecting tubing. Slow time constants of detectors and recorders may also contribute. These extra-column effects are more severe for early, narrow peaks in the chromatogram than for later, broader peaks. A more detailed discussion of these effects will be given in the section on instrumentation. 

The volume of the column available to the mobile phase or the mobile phase volume (also called dead volume) is dependent on the geometry of the column and the packing. A contribution to the mobile phase volume will also be made by the injection system, by the connecting tubing and by the detector. Thus the contributions may be separated into in-column and extra-column effects. 

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