Mass loadability

Mass loadability of SPE and RAM columns play a key role in executing the sample clean-up. It is advisable to work below the overload regime of the column. Otherwise, displacement effects and other phenomena such as secondary interaction by adsorbed species might take place, which will lead to nonrepro-ducible results. This last statement is particularly important when the task is to monitor medium-to-low-abundance proteins large sample volumes in the milliliter range are therefore usually applied. 

The primary criteria for the choice of a separation phase system are selectivity and orthogonality, mass loadability, and biocompatibility (in case of quantification). 

As a rule of thumb, the first dimension should possess a high mass loadability combined with sufficient selectivity and maintenance of bioactivity. Ion exchange chromatography (lEC) is therefore the method of choice offering charge selectivity. 

Column miniaturization from millimeters to micrometers I. D. has two consequences apart from flow-rate reduchon the injection volume decreases as well as the mass loadability. The diminuhon of flow-rate and injection volume in relation to column I. D. and an example of the mass loadability for peptides of RP columns of gradated I. D. is displayed in Table 2. 

Table 5.2 Estimation of HPLC column mass loadability.

Column 1. D. (pm) Flow rate (pL min" ) Column volume (hQ Mass of silica per column %) Mass loadability per column (hs)... 

The optimum average particle size of preparative stationary phases with respect to pressure drop, plate number and mass loadability is between 10 and 15 p,m. 

Gas sorption (nitrogen at 77 K), mercury intrusion (mercury porosimetry) Specific surface area (BET), pore size distribution, average pore diameter, specific pore volume, particle porosity Retention of solutes, mass loadability, column regeneration, column performance, mass loadability, pore and surface accessibility for solutes of given molecular weight, mechanical stability, column pressure drop, pore connectivity... 

All these parameters depend on the mass loadability of the column and change significantly when a critical loadability is reached. The critical mass loadability of analytical columns is usually reached at a 10% reduction of the retention coefficient or at 50 % decrease of column plate number. At higher values the column is, in chromatographic terms, overloaded. 

Estimates of the loading capacity of a particular column material can be usually obtained from the manufacturer. The mass loadability for a scaled up separation can be calculated with the following formula ... 

All these parameters depend on the mass loadability of the column and change significantly when a critical loadability is reached. The critical mass... 

Figm 15.7 Comparison of a measured and a calculated mass loadability curve. (The data were obtained from Ref. 4.)... 

As we have seen at the beginning of the previous section, there is a difference of nearly 4 orders of magnitude between the external and the internal surface of a 5 on fully porous particle with a pore size of around lOrun. If we compare a 1-pm nonporous particle to a fiilly porous particle, the difference in surface area per column volume is still about 3 orders of magnitude. This means that the mass loadability of a packed bed with a l-/im nonporous particle is 3 orders of magnitude lower than that of the fully porous partide with 10-mn pores. 

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