Sample Mass Overload

Band dispersion from sample mass overload is a direct result of the chromatographic process proceeding under conditions, where the adsorption isotherm of the solute on the stationary phase, is no longer linear. The development of an equation that describes the extent, of band spreadinn as a function of mass of sample placed on the column, is complex. This problem has been elegantly approached by 6uiochon and his co-workers (15-18) from the basis of the adsorption isotherm of the solute on the stationary phase. 

The form of the isotherm need not be Langmuir in nature, but in any event, must be experimentally determined in order to identify the true profile of the overloaded peak. In practice, the determination of the adsorption isotherm of each compound to be separated by a preparative chromatographic procedure can be arduous and time consuming. A better alternative might be to design the fully optimized column from basic principles in the manner previously described. 

Knox and Pyper (14) assumed that the majority of the adsorption isotherms were, indeed, Langmuir in form and then postulated that all the peaks that were overloaded would be approximately triangular in shape As a consequence, Knox and Pyper assumed that mass overload could be treated in a similar manner to volume overload. Whether all solute/stationary phase isotherms are Langmuir in type, is a moot point, and the assumption should be taken with some caution. Knox and Pyper then suggested that the best compromise was to utilize about half the maximum sample volume as defined by equation (15) which would then reduce the distance between the peaks by half. They then recommended that the concentration of the solute was increased until dispersion due to mass overload just caused the two peaks to touch. Knox summarized his recommendations in the following way (19), 

1/ Develop an analytical separation which gives the best possible resolution between the critical solutes. 

2/ Determine the difference between the retention volumes of the two solutes (AV) (equation (15)). 

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Preparative Chiral Chromatography The Loading Capacity of a Column The Maximum Sample Volume Sample Volume Overload Sample Mass Overload Preparative Chromatography Apparatus Solvent Reservoirs Pumps... 

The major cause of peak asymmetry in GC is sample overload and this occurs mostly in preparative and semi-preparative separations. There are two forms of sample overload, volume overload and mass overload. 

Volume overload results from too large a volume of sample being placed on the column, and this effect will be discussed later. It will be seen that volume overload does not, in itself, produce asymmetric peaks unless accompanied by mass overload, but it does broaden the peak. Mass overload, however, frequently results in a nonlinear adsorption isotherm. However, the isotherm is quite different from the Langmuir isotherm and is caused by an entirely different phenomenon. 

The problem is made more difficult because these different dispersion processes are interactive and the extent to which one process affects the peak shape is modified by the presence of another. It follows if the processes that causes dispersion in mass overload are not random, but interactive, the normal procedures for mathematically analyzing peak dispersion can not be applied. These complex interacting effects can, however, be demonstrated experimentally, if not by rigorous theoretical treatment, and examples of mass overload were included in the work of Scott and Kucera [1]. The authors employed the same chromatographic system that they used to examine volume overload, but they employed two mobile phases of different polarity. In the first experiments, the mobile phase n-heptane was used and the sample volume was kept constant at 200 pi. The masses of naphthalene and anthracene were kept... 

To demonstrate the effect in more detail a series of experiments was carried out similar to that of volume overload, but in this case, the sample mass was increased in small increments. The retention distance of the front and the back of each peak was measured at the nominal points of inflection (0.6065 of the peak height) and the curves relating the retention data produced to the mass of sample added are shown in Figure 7. In Figure 7 the change in retention time with sample load is more obvious the maximum effect was to reduce the retention time of anthracene and the minimum effect was to the overloaded solute itself, benzene. Despite the reduction in retention time, the band width of anthracene is still little effected by the overloaded benzene. There is, however, a significant increase in the width of the naphthalene peak which... 

The technique of column overloading is only feasible if more than adequate resolution is possible between the solute of interest and its nearest neighbor. Many samples require a column to be constructed that will only just separate the solutes of interest and under these circumstances the loading capacity must be increased without overloading the column. It has been shown in earlier chapters that the maximum sample (mass or volume) that can be placed on a column is proportional to the plate volume of the column and the square root of its efficiency. Thus, the maximum sample mass (M) will be given by... 

Preparative chromatography involves the collection of individual solutes as they are eluted from the column for further use, but does not necessarily entail the separation of large samples. Special columns can be designed and fabricated for preparative use, but for small samples the analytical column can often be overloaded for preparative purposes. Columns can be either volume overloaded or mass overloaded. Volume overload causes the peak to broaden, but the retention time of the front of the peak... 

From a practical point of view it is better to use the maximum volume of sample possible, assuming there is no mass overload. This will allow the detector to be operated at the lowest possible sensitivity and in doing so provide the greatest detector stability and, as a consequence, the highest accuracy. 

The easiest way for an analyst to obtain small quantities of a component of a mixture is to overload an analytical column. In order to exercise this technique, the solute of interest must be well separated from its closest neighbor. The column can then be overloaded with sample until the peak dispersion resulting from the overload, causes the two peaks to touch at their base. There are two types of column overload, volume overload and mass overload. In practice, it is often advantageous to employ a combination of both methods and a simple procedure for doing this will be given overleaf. 

Volume overload can be treated in a simple way by the plate theory (8,9). In contrast, the theory of mass overload is complicated (10-12) and requires a considerable amount of basic physical chemical data, such as the adsorption isotherms of the solutes, before it can be applied to a practical problem. Volume overload is useful where the solutes of interest are relatively insoluble in the mobile phase and thus, to apply a sample of sufficient size onto the column, a large sample volume is necessary. If the sample is very soluble in the mobile phase then mass overload might be appropriate. 

Volume overload employing a solution of the material in the mobile phase at a level of about 5% w/v is a recommended method of sampling for preparative columns if the system is not optimized. However, a combination of volume overload and mass overload has also been suggested as an alternative procedure by Knox (13). 

Finally, an officially updated definition of the retardation factor, R, issued by lUPAC is important to the whole field of planar chromatography (the linear and the nonlinear TLC mode included). The importance of such a definition has two reasons. First, it is promoted by the growing access of planar chromatography users for densitometric evaluation of their chromatograms and second, by the vagueness of the present definition in the case of skewed concentration profiles with the samples developed under mass overload conditions. 

Overloading is a term that describes the decrease in efficiency or change in retention that occurs with increased quantities of sample it can be caused by excessive sample volume or sample mass [7]. It results from the nonlinearity of the distribution of the... 

In practice the value of (w) will vary between about 2 and 5 ( i.e sample concentrations will lie between 2%w/v and 5%w/v) before mass overload becomes a significant factor In band dispersion. A numerical value for (g>) of 5 will be taken In subsequent calculations. The correct value of ( ), for the particular solute concerned, can be experimentally determined on an analytical column carrying the same phase system If so required. 

For any given chromatographic system, there is a limiting charge that can be placed on a column before the resolution is impaired. Loss of resolution from column overload can arise from two causes, either excessive sample feed volume or excessive sample mass. The theory of moderate sample volume overload has already been considered in the applications of the Plate Theory. The theory of excessive sample volume overload will now be discussed. 

Often a large sample injection (e.g., 200 juJL) will experience only a little additional band broadening over that which would occur for a small injection (e.g., 10 (jlL). Thus, the large injection can result in a higher concentration for detection with no loss in resolution. (This assumes there is no mass overload, which is often a valid assumption in situations where insufficient sensitivity occurs due to very small concentrations of the sought-after substance. However, experimentally this assumption should be verified for each situation.) Also, improved chromatographic precision can result if there is smaller uncertainty of measurement with the larger volume injection. 

Mass overload occurs when the stationary phase does not have the capacity to retain the amount of sample injected. This can occur even for small injection volumes if the concentration of sample is high enough. This results in a characteristic shark-fin peak shape, where peak tailing starts from the peak s apex. For example, in order to obtain sufficient sensitivity, analytes with weak UV molar absorptivity may require a large enough amount of sample to be injected that the stationary phase becomes overloaded. Injecting less amount of sample, either by a smaller injection volume or by diluting the sample, can solve the problem of mass overload. However, sensitivity will decrease in this case. 

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