Preparative mass overload

A chromatographic separation can be developed in three ways, by displacement development, by frontal analysis, and by elution development, the last being almost universally used in all analytical chromatography. Nevertheless, for the sake of completeness, and because in preparative chromatography (under certain conditions of mass overload) displacement effects occur to varying extents, all three development processes will be described. 

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. 

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. 

Figure 2.23 Mass overload. Top 2 mg each of acetophenone (first peak) and veratrole bottom 2ixg each. Column, 25cmx3.2mm i.d. stationary phase, LiChrosorb SI 60 5ixm mobile phase, hexane-diethyl ether 9 1, 1 ml min UV detector, 290 nm, with preparative or analytical cell, respectively.

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. 

Due to the complex interactions, simple calculations as can be performed for single solutes are of no - or very little - value in multi-component preparative chromatography. All results which have led to our better understanding of mass overload have arisen from a combination of experiment and computer simulation. The key to... 

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... 

In cases where mass transfer is rapid, as is the case with most small molecule separations, then isocratic elution can offer advantages such as automatic fraction reprocessing and solvent recycle. However, with larger synthetic objectives the rate of mass transfer is comparatively low so isocratic elution leads to band broadening and subsequently to recovery of the peptide at high dilution. Most preparative HPLC based peptide separations are carried out under gradient and overload conditions that allow for maximum throughput in terms of time and quantity. 

---