Extra-column contributions to band broadening

Relationships between mobile phase velocity and column efficiency, such as the Knox equation (equation (2.32)), are developed by taking into account all the possible contributions to band broadening. Thus the total peak variance (a oiumn) arising from band broadening in a packed column is given by 

Theoretically, the injection volume (F nj) of a sample should be substantially less than the peak volume. However, in practice very large injection volumes that are well in excess of the peak volume can be made provided the injection solvent is much weaker than the mobile phase used for elution. This is possible because of zone compression, which causes a sample injected in a weak solvent to be compressed at the head of the column. Zone compression can usually be achieved if the concentration of organic modifier in the sample solution is less than 50% of the concentration of the organic modifier in the mobile phase. Even if the sample is dissolved in the mobile phase, substantial zone compression will occur if the chromatographic conditions are arranged such that k 5. 

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Although the above calculation is somewhat oversimplified because the effects of the compressibility of the gas have been neglected, it serves to illustrate that a reduction of the column diameter cannot be fully compensated by an increase in the column length to keep the column dispersion constant. Therefore, when narrow-bore capillary columns are to be used in GC, the extra-column contribution to band broadening will need to be reduced. 

Pellicular or controlled surface porosity particles were introduced in the late 1960s these have a solid inert impervious spherical core with a thin outer layer of interactive stationary phase, 1-2 pm thick [13]. Originally, the inner sphere was a glass bead, 35-50 pm i.d., with a thin active polymer film or a layer of sintered modified silica particles on its surface. Such particles were not very stable, had very low sample load capacities because of low surface areas and are not used any more. Nowadays, this type of material is available as micropellicular silica or polymer-based particles of size 1.5 to 2.5 pm [14]. Micropellicular particles are usually packed in short columns and because of fast mass-transfer kinetics have outstanding efficiency for the separation of macromolecules. Because the solutes are eluted as very sharp narrow peaks, such columns require a chromatograph designed to minimise the extra-column contributions to band broadening. 

The main disadvantage of size-exclusion chromatography is its low peak capacity, which arises from the small elution volumes of the peaks. In addition because peak dispersion is small, extra-column contributions to band broadening in size-exclusion chromatography have to be kept to a minimum. The relative contribution of extra-column band broadening can be minimized by the use of small particles = 3-10 pm) and long (L = 50 100 cm), wide-bore (d = 8-10 mm) columns. 

Care must be taken to keep additional band-broadening outside the column to a minimum. Extra-column contributions to band broadening, such as mixing or poor sample-introduction technique, will increase the //-value. Because of the slow diffusion of samples in liquid phases, extra-column volumes are more detrimental to efficiency in LC than in GC hence, great effort should be exercised to keep those volumes between the point of injection and the top of the column to a minimum. Likewise, the volume between the column exit and the detector, and the volume of the detector itself, should be minimized. In TLC or PC, the applied spot should be kept as small as possible. 

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. 

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 ... 

Extra-column volumes should be kept as low as possible because they affect the separation. They are one cause of band broadening and of tailing. Their contribution to band broadening is additive. If each part adds 5% to peak width the decrease in separation performance is not negligible at all. Whereas it can be difficult to modify the instrument with regard to injector and detector, it is possible to use adequate tubing to connect the parts. Adequate means capillaries with an inner diameter of 0.17 or... 

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 detector time constant can distort column efficiency when the peak width (in time units) becomes of the same order of magnitude as the response time. High-efficiency columns produce very sharp peaks, and detectors with response times greater than 0.5 s can contribute significantly to band broadening. Electronic filtering can increase response time and cause measurable broadening of sharp peaks. Refer to Reference 109 for an exhaustive discussion of extra-column effects in detector systems. 

The detector flow-cell, the contribution of which to ctv is approximately equal to its volume [707], represents a considerable and recognizable contribution to the extra-column band broadening. Typical conventional flow-cells have a volume of 8 pi, which is quite substantial compared with the maximum allowable extra-column dispersion. 

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. 

In order to obtain the characteristics of the column the values of and must be first corrected for the extra-column band broadening contributions that... 

Since the model given by Eq. 6.133 concerns only the stationary phase process, the mobile phase dispersion and extra column broadening effects have to be corrected for via the deconvolution of the whole chromatogram with the peak of the imretained marker that contains the necessary information regarding these contributions to the band profiles. The resulting net chromatogram carries the contribution of the stationary phase processes only. 

The contribution of connecting tubing to extra-column effects has been already discussed in the literature. All parameters have been studied experimentally to obtain a mathematical relationship describing their mutual dependence. Such an equation is given below, where the band broadening in a chromatographic system is considered to be inversely affected by the flow rate... 

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. 

With modem GC instrumentation, the extra peak variance contribution (electrometer, data system, recording) approaches zero. Because of the occuneiice of the squared variances in (his equation, the contribution of the initial bandwidth to the total peak bandwidth is less dramatic than might be expected, at least for GC on packed columns. For capillary columns, injection band-widths can be too large compared to the chromatographic band broadening, and narrow initial bands have to be obtained by ... 

To achieve maximum performance for a column, extra-column band-broadening in Eq. (3.6) must be minimized. There are basically two types of extra-colunm contributions. The primary one is volumetric in nature and originates from the injection volume, the detector volume, and the volume of connection tubing between the injector and detector. The other type of band-broadening derives from time-related events, such as the sampling rate and the detector time constant. For modem detectors and data acquisition software, the latter contribution can be negligible when fast data acquisition speed and low detector time constants are used. 

Primary contributions to extra-column band-broadening in Eq. (3.6) are from the injection volume, connection tubing, and the flow cell. Under given chromatographic conditions, the variance of a sample plug from a connection tubing is described by the Taylor-Aris Eq. (3.1) ... 

Equation (3.6) indicates that the observed or apparent band-broadening arises from two sources the column itself and the extra-column volume. The relative contribution of these two sources to extra-band-broadening is primarily related to the retention factor of an analyte, the column internal diameter, and the extra-column volume if other factors are kept constant. 

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