Unavailable stationary phase

Packed columns coated with a wide variety of stationary phases are commercially available. Unlike open tubular columns they are easily prepared in the laboratory and many chromatographers prefer to prepare their own columns. Almost any liquid not restricted by its vapor pressure can be studied extending the scope of packed columns to stationary phases unavailable on open tubular columns as well as allowing gas chromatography to be used to obtain physicochemical properties of a wide range of materials evaluated as stationary phases. 

Recalling the plate theory, it must be emphasized that (Vm) is not the same as (Vm)-(Vm) is the moving phase and a significant amount of (Vm) will be static (e.g., that contained in the pores). It should also be pointed out that the same applies to the volume of stationary phase, (Vs), which is not the same as (Vs), which may include material that is unavailable to the solute due to exclusion. 

The stationary phase can be apportioned in a similar manner. For example, with a bonded phase, due to the porous nature of the support, some of the pores will become blocked with stationary phase and so the total amount of stationary phase can be divided into that which is chromatographically available (Vs(A)) and that which is chromatographically unavailable (Vs(u)). 

Volume of Chromatographically Unavailable Stationary Phase (by diff.) - total mobile phase in the column,... 

It is now necessary to consider the volume of stationary phase in the column. Owing to the nature of the majority of bonded phases and as a result of their method of manufacture, some of the smaller pores may become completely blocked by the stationary phase itself, and thus some stationary phase may become chromatographically unavailable. 

Volume of Chromatographically Unavailable Stationary Phase (by difference)... 

Larger molecules such as proteins usually do not fit these predictions, probably because the molecules adopt an ordered three-dimensional structure in which many of the hydrophobic residues are buried within the structure and unavailable for interaction with the reversed phase. As might be expected from the proposed mechanism of separation, the retention of proteins on reversed-phase columns is not related to molecular weight of the sample, but rather the surface polarity of the molecule. Table I shows that there is a correlation of hydrophobicity (measured by mole % of strongly hydrophobic residues) with retention order for seven different proteins. It is unlikely that the retention of all proteins on a reversed-phase column can be correlated in this manner, because many protein structures have few nonpolar residues exposed to the aqueous environment. For example, although the major A and C apolipoproteins are eluted from a ju-Bondapak alkylphenyl column in an order which can be related to the proposed secondary structures, there is little correlation with the content of hydrophobic residues in each protein and the degree of interaction with the stationary phase. A similar lack of correlation be-... 

The mobile phase plays a number of interactive roles in a chromatographic separation. First and foremost, it is in dynamic equilibrium with the stationary phase. For base silica (and other unmodified supports), there exists an adsorbed layer of mobile phase components, not necessarily identical to the mobile phase composition. This is why water deactivates a silica column it forms strong hydrogen bonds with the residual silanol groups (the active adsorption sites on the surface) and renders them unavailable for adsorptive interactions with the solute. 

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