Volume sample overload

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. 

Consider the situation depicted in figure 7. To determine the band dispersion that results from a significant, but moderate, sample volume overload the principle of the summation of variances can be applied. However, when the sample volume becomes excessive, the band dispersion that results from the overload becomes equivalent, to a first approximation, to the sample volume itself. In figure 6, two solutes are depicted that are eluted from a column 

Two Solutes Eluted Under Conditions of No Volume Overload 

Noting also that (n) is large, and thus, to the first approximation, 

It is seen from equation (15) that the maximum overload volume is linearly related to the (k ) value of the first eluted solute of the critical pair, the function (a-1) and the column dead volume, Consequently, the larger the column, either in length and/ or radius, the larger the sample volume can be, This assumes that the column is of such a size, that it can be efficiently packed with practical techniques and that the particle size of the packing is chosen such that the pump pressure available can provide the necessary mobile phase flow-rate. 

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To determine the band dispersion that results from a significant, but moderate, sample volume overload the summation of variances can be used. However, when the sample volume becomes excessive, the band dispersion that results becomes equivalent to the sample volume itself. In figure 10, two solutes are depicted that are eluted from a column under conditions of no overload. If the dispersion from the excessive sample volume just allows the peaks to touch at the base, the peak separation in milliliters of mobile phase passed through the column will be equivalent to the sample volume (Vi) plus half the base width of both peaks. It is assumed in figure 10 that the efficiency of each peak is the same and in most cases this will be true. If there is some significant difference, an average value of the efficiencies of the two peaks can be taken. 

Sample volume overloading can also reduce efficiency—for example, when the sample plug length is wider than the zone length determined by diffusion. This liability of high injectable volumes is often ignored, and efficiency is intentionally sacrificed to gain sensitivity. 

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... 

Hgure 1 Preparative LC peak shapes resulting from (A) sample concentration overload and (B) sample volume overload. 

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. 

If the sample is relatively insoluble in the mobile phase, then it can be dissolved, as a dilute solution, in a relatively large volume of solvent. A large volume of the solution can then be placed on the column, a procedure that results in volume overload. 

Consider the separation depicted in Figure 1. It is assumed that the pair of solutes represent the elution of the solute of interest and its nearest neighbor. Now, when the sample volume becomes extreme, the dispersion that results from column overload, to the first approximation, becomes equivalent to the sample volume itself as the sample volume now contributes to the elution of the solutes. Thus, from Figure 1, the peak separation in milliliters of mobile phase will be equivalent to the volume of sample plus half the sum of the base widths of the respective peaks. 

The effective use of column volume overload for preparative separations was experimentally demonstrated by Scott and Kucera [1]. These authors used a column 25 cm long, 4.6 mm I.D. packed with Partisil silica gel 10 mm particle diameter and employed n-heptane as the mobile phase. The total mass of sample injected was kept constant at 176 mg, 8 mg and 0.3 mg of benzene, naphthalene and anthracene, respectively, but the sample volumes used which contained the same mixture of solutes were 1 pi, 1 ml, 2 ml and 3 ml. The chromatograms of each separation are... 

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... 

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... 

Errors in the molecular weight data from HPSEC are usually due to improperly prepared samples, column dispersity, or flow rate variations. The sample to be analyzed should be completely dissolved in the mobile phase and filtered prior to injection onto the column. A plugged column inlet frit will invalidate results. In addition, do not load the column with excess sample. Column overloading affects the accuracy of data by broadening peaks, reducing resolution, and increasing elution volume. For best results, the concentration of the injected sample should be as low as possible while still providing adequate... 

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). 

Knox and Piper (13) assumed that the majority of the adsorption isotherms were, indeed, Langmuir in form and then postulated that all the peaks that were mass overloaded would be approximately triangular in shape. As a consequence, Knox and Piper proposed 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 Piper 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 should be increased until dispersion due to mass overload just caused the two peaks to touch. 

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... 

As long as the boundary and initial conditions remain unchanged, the band profiles on the reduced time and length scale depend only on the column efficiency. The conventional boundary and initial conditions for all modes of chromatography state that (1) the column is equilibrated with the mobile phase prior to the beginning of the separation (2) the sample is then injected as a rectangular pulse and (3) the separation proceeds as required by the specific mode selected. The amount of sample injected is determined by the volume and the concentration of the feed injected. As long as we avoid serious volume overload, the actual values of these two parameters are immaterial. Only their product, i.e., the amount injected, will influence the band profile. 

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