Chromatographic modes, column types

When the chromatographic mode, column type, packing and dimensions have been chosen, the final stage of method development involves solvent optimisation and a choice between isocratic or gradient elution. Many separations can be achieved perfectly satisfactorily under isocratic conditions and are preferred to gradient elution techniques, as these are inconvenient due to the time required to re-equilibrate the column. A measure of the quality of separation is given by the resolution factor which can be expressed as follows ... 

There are two classes of stationary phases in SEC, one type for GFC and the other for GPC. Stationary phases for GFC are hydrophilic and include polydextrans, polyvinyl alcohol gel, and silica gel those for GPC are hydrophobic, typically cross-linked, rigid polystyrene-divinylbenzene gels. Generally, columns of 15 to 50 cm length are used, packed with 7- to 10-/t,m particles and with an internal diameter between 0.6 and 0.8 cm. In SEC, unlike in other chromatographic modes, the stationary phase is the primary factor controlling retention. 

The injector, which is the sample s entrance to the chromatograph, has different functions. Besides its role as an inlet for the sample, it must vaporize, mix with the carrier gas and bring about the sample at the head of the column. The characteristics of the injectors, as well as the modes of injection, differ according to column type. The use of an automatic injection system can significantly enhance measurement precision. 

Equilibration times vary widely with the type of chromatography and the history of the column. A colunm that had previously been equilibrated and was stored in the mobile phase should be up and running within a few, maybe 10, column volumes, provided the new mobile phase is really identical to the old mobile phase. If the column was stored in a solvent other than the mobile phase, equilibration times vary with the chromatographic mode. Normal-phase chromatography using silica or alumina columns may take a long time for equilibration several days at a flow rate of 1 column volume per minute have bran observed. The equilibration with normal-phase bonded phases is faster. 

With notable exceptions, the application of HPLC to clinical chemistry has not as yet been extensive. This is somewhat surprising in view of the potential the method has for this area. This potential arises, in part, from the fact that HPLC is well suited to the types of substances that must be analyzed in the biomedical field. Ionic, relatively polar species can be directly chromatographed, without the need to make volatile derivatives as in gas chromatography. Typically, columns are operated at room temperature so that thermally labile substances can be separated. Finally, certain modes of HPLC allow fractionation of high molecular weight species, such as biopolymers. 

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