Overload, column volume

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... 

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. 

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... 

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. 

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. 

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). 

In 2001, Valko et al. reduced the column length to only 50 mm and increased the flow rate to 2mLmin [42]. The gradient time was diminished to 2.5 min with a gradient cycle time of 5 min. Measurement of CHI and evaluation of log P were excellent with a 3-fold improved productivity. In these conditions, the system dwell volume (Vd) becomes essential and only dedicated chromatographic devices with Vjy lower than 0.8 mL can be used [42]. Special attention should be paid to the injected volume, which must remain lower than 3 pL to avoid any overloading or extra-column volume contributions. 

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. 

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. 

Due to small particle size, the columns packed with micropellicular stationary phases have low permeability (27) and therefore, can not be operated at very high flow rates due to pressure limitations of commercial HPLC instruments. In comparison to porous particles, the surface area of stationary phases per unit column volume is low, and hence, their loading capacity is correspondingly smaller. This is particularly evident in the isocratic analysis of small molecules where the column can be easily overloaded. Therefore, micropellicular sorbents do not appear to offer advantages in the HPLC of small molecules. 

The injection is a critical factor in fast LC methods and must be considered to maintain column efficiency. Injection volumes that are too large can cause volume overload of the column, which results in broad, flat-top shaped peaks with low plate counts that are more pronounced for earlier eluting components. As injection volume is increased, peak height should increase however, peak width should remain the same. If peak width increases as well, this is indicative of volume overload. As column dimensions are reduced, the maximum injection volume must be reduced by the ratio of the column volumes [see equation (17-33) in Section 17.7.4], For example, reducing... 

Alternatively, QAE-Sephadex A-50 may be rapidly equilibrated with ethylene-diamine (2.88 g/l)-acetic acid (73 ml of 1 M/1) buffer, pH 7.0, in a fume hood. The rabbit or human serum is diluted with an equal volume of the same buffer, and applied to the column. IgG passes through while other proteins are adsorbed. Contaminants may be desorbed with a buffer consisting of 435 ml 600 mM acetic acid and 130 ml 600 mM sodium acetate per litre (pH 4.0). Volume change of the ion-exchanger is avoided since the ionic strength of the buffer is maintained at 0.1 (Tijssen and Kurstak, 1974). The yield is 70-85% for the various sera with at least 90% purity if overloading is avoided. Not more than half the column volume of diluted serum should be applied. 

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