Preparative 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. 

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... 

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... 

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). 

The major cause of peak asymmetry in LC is sample overload and this occurs mostly in preparative and semi preparative LC. 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 over load does not, in itself, produce asymmetric peaks unless accompanied by mass overload. Mass overload which, as discussed above, is accompanied by a distortion of the normally linear isotherm, can cause very significant peak asymmetry and, in fact, seriously impair the resolution obtained from the column. 

The user of preparative HPLC in general wants to obtain as much of a pure compound per unit time as possible. Therefore, it is necessary to work under conditions of overload. If sample solutions are diluted, volume overload will... 

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 user of preparative HPLC in general wants to obtain as much of a pure compound per unit time as possible. Therefore, it is necessary to work under conditions of overload. If sample solutions are diluted, volume overload will preferentially occur whereas mass overload is common with concentrated samples. Often both effects are present and the peaks become truncated, as can be seen at the bottom of Fig. 20.3 (with increasing retention the plateau is lost and the peaks become triangular). The maximum possible injected amount of a concentrated solution is determined empirically the injection volume is increased until the peaks touch each other. Non-diluted samples are not suitable. 

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

The sample concentration which corresponds to the volume overload example above is approximately 0.3 mg/mL Normally a sample solubility of between 10 and lOOmg/mL is desirable for preparative separations. A very poor solubility such as this usually is unacceptable and changes in temperature, mobile phase or the entire phase system are made in order to improve it to the level where mass effects predominate. There is a further need to reduce the volume overload effects other than those noted here. This is a consequence of the interactions between the solutes, which are considered in the following section. 

The final purification steps are responsible for the removal of the last traces of impurities. The volume reduction in the earlier stages of the separation train are necessarv to ensure that these high-resolution operations are not overloaded. Generally, chromatograjmy is used in these final stages. Electrophoresis can also be used, but since it is rarely found in process-scale operations, it is not addressed here. The final product preparation may require removal of solvent and drying, or lyophihzation, of the product. 

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... 

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