Stationary and mobile phases

The sample components (solutes) can interact directly with components of the mobile phase, except in gas chromatography where there are no such interactions and the mobile phase is simply a carrier gas for the sample components. 

When the stationary phase is a solid, often with polar surface groups, and the mobile phase is either a gas (in GC) or an organic solvent (in LC), the separation principle is based on adsorption, and the term adsorption chromatography can be used. Other not so commonly used terms are gas-solid chromatography and liquid-solid chromatography. The adsorption forces include dispersion interactions, dipolar interactions, add-base interactions, complexation, and so on. 

In gas chromatography, the stationary phase can also be a hquid, where the separation prindple is based on partition between the two phases. This was also the case formerly in liquid chromatography, but after the introduction of chemically bonded stationary phases into HPLC, the stationary phase cannot be described as a liquid anymore. 

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A separation in which solutes partition between a mobile and stationary phase. 

Chromatographic separations are accomplished by continuously passing one sample-free phase, called a mobile phase, over a second sample-free phase that remains fixed, or stationary. The sample is injected, or placed, into the mobile phase. As it moves with the mobile phase, the sample s components partition themselves between the mobile and stationary phases. Those components whose distribution ratio favors the stationary phase require a longer time to pass through the system. Given sufficient time, and sufficient stationary and mobile phase, solutes with similar distribution ratios can be separated. 

A quantitative means of evaluating column efficiency that treats the column as though it consists of a series of small zones, or plates, in which partitioning between the mobile and stationary phases occurs. 

In gas chromatography (GC) the sample, which may be a gas or liquid, is injected into a stream of an inert gaseous mobile phase (often called the carrier gas). The sample is carried through a packed or capillary column where the sample s components separate based on their ability to distribute themselves between the mobile and stationary phases. A schematic diagram of a typical gas chromatograph is shown in Figure 12.16. 

Reversed-phase columns are used to separate polar substances. Although in LC the stationary phase is a solid, it is necessary to bear in mind that there may be a thin film of liquid (e.g water) held on its surface, and this film will modify the behavior of sample components equilibrating between the mobile and stationary phases. A textbook on LC should be consulted for deeper discussion on such aspects. 

Distribution Coefficients. Gel-permeation stationary-phase chromatography normally exhibits symmetrical (Gaussian) peaks because the partitioning of the solute between mobile and stationary phases is linear. Criteria more sophisticated than those represented in Figure 8 are seldom used (34). 

An advantage of Hquid chromatography is that the composition of the mobile phase, and perhaps of the stationary phase, can be varied during the experiment to provide greater efficacy of the separation. There are many more combinations of mobile and stationary phases to effect a separation in Ic than one would have in a similar gas chromatographic experiment, where the gaseous mobile phase often serves as Httle more than a convenient carrier for the components of the sample. 

The concentration profiles of the solute in both the mobile and stationary phases are depicted as Gaussian in form. In due course, this assumption will be shown to be the ideal elution curve as predicted by the Plate Theory. Equilibrium occurs between the mobile phase and the stationary phase, when the probability of a solute molecule striking the boundary and entering the stationary phase is the same as the probability of a solute molecule randomly acquiring sufficient kinetic energy to leave the stationary phase and enter the mobile phase. The distribution system is continuously thermodynamically driven toward equilibrium. However, the moving phase will continuously displace the concentration profile of the solute in the mobile phase forward, relative to that in the stationary phase. This displacement, in a grossly... 

High-performance liquid chromatography is in some respects more versatile than gas chromatography since (a) it is not limited to volatile and thermally stable samples, and (b) the choice of mobile and stationary phases is wider. 

The profile of the concentration of a solute in both the mobile and stationary phases is Gaussian in form and this will be shown to be true when dealing later with basic chromatography column theory. Thus, the flow of mobile phase will slightly displace the concentration profile of the solute in the mobile phase relative to that in the stationary phase the displacement depicted in figure 1 is grossly exaggerated to demonstrate this effect. It is seen that, as a result of this displacement, the concentration of solute in the mobile phase at the front of the peak exceeds the equilibrium concentration with respect to that in the stationary phase. It follows that there is a net transfer of solute from the mobile phase in the front part of the peak to the... 

The two components which are associated with the separation that occurs in a chromatographic system are the mobile and stationary phases. 

Unlike gas chromatography, in which the mobile phase, i.e. the carrier gas, plays no part in the separation mechanism, in HPLC it is the relative interaction of an analyte with both the mobile and stationary phases that determines its retention characteristics. Hence, it is the varying degrees of interaction of different analytes with the mobile and stationary phases which determines whether or not they will be separated by a particular HPLC system. 

If the identity of the analyte is genuinely unknown, a farther problem is encountered. In contrast to GC, there are no HPLC systems, combinations of mobile and stationary phases, that are rontinely used for general analyses. Therefore, there are no large collections of k values that can be nsed. For this reason, retention characteristics are often nsed for identification after the nnmber of possible compounds to be considered has been greatly reduced in some way, e.g. the class of compound involved has been determined by colonr tests or UV spectroscopy. 

The selectivity (separation capability) of an HPLC system is dependent upon the combination of mobile and stationary phases. Since ions are being generated directly from the mobile phase by electrospray, its composition, including the identity and concentration of any buffer used, and its flow rate are important considerations. 

Theoretical plate In plate theory, the chromatographic column is viewed as a series of narrow layers, known as theoretical plates, within each of which equilibration of the analyte between mobile and stationary phases occurs. 

Other modes of LC operation include liquid-liquid partition chromatography (LLC) and bonded phase chromatography. In the former, a stationary liquid phase which is immiscible with the mobile phase is coated on a porous support, with separation based on partition equilibrium differences of components between the two liquid phases. This mode offers an alternative to ion exchange in the fractionation of polar, water soluble substances. While quite useful, the danger exists in LLC that the stationary phase can be stripped from the column, if proper precautions are not taken. Hence, it is typical to pre-equil-ibrate carefully the mobile and stationary phases and to use a forecolimn, heavily loaded with stationary phase 9). 

The precision in retention from injection to injection will often be better than 1%. Over longer periods of time such precision requires the following (a) good flow control from the pump (b) constant mobile and stationary phases and (c) temperature control of the column. The critical question of reproducibility from column to column is still a matter of concern, especially when dealing with the more sophisticated packing materials, e.g. small porous particles, bonded phases While frequently this reproducibility is quite good, workers should recognize that care must be exercised to achieve and/or maintain reproducible columns. Undoubtedly, with experience, this need not be a severe problem. 

Solvent selectivity is seen as the factor that distinguishes individual solvents that have solvent strengths suitable for separation. In reality, separations result from the competition between the mobile and stationary phases for solutes based on the differences of all intermolecular interactions with the solute in both phases. Solvents can be organized on selectivity scales that are useful for initial solvent selection, but in a chromatographic separation the properties of the stationary phase must be taken into consideration. Methods that attempt to model chromatographic separation need to consider simultaneously mobile and stationary phase properties [38]. 

In its turn, PLC can be used as a pilot technique for column preparative chromatography with the same system of mobile and stationary phases. 

The A term represents the contribution from eddy diffusion, the B term the contribution from longitudinal diffusion, and the C terms the contributions from mass transfer in the mobile and stationary phases to the total column plate height. By differentiating equation (1.31) with respect to the mobile phase velocity and setting the result equal to zero, the optimum values of mobile phase velocity (u ) and plate height (HETP ) can be obtained. 

Figure 4.17 General phenonenaloglcal retention model for a solute that participates in a secondary chemical equilibrium in liquid chromatography. A - solute, X - equilibrant, AX analyte-equilibrant coeplex, Kjq - secondary chemical equilibrium constant, and and are the primary distribution constants for A and AX, respectively, between the mobile and stationary phases.

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