Stationary phase functionalities

This mode of separation, as the name suggests, uses stationary phases with a special affinity for a specific analyte. The affinity ligand immobilized on the stationary phase varies dramatically from peptide, to protein, to oligonucleotide, to monoclonal antibody. In some cases the target molecule is labelled with an affinity tag to simplify the separation. This approach is common in the synthesis of recombinant proteins where the system can be engineered so that the target biomolecule expresses a tag such as polyhistidine. A stationary phase functionalized with aminodiacetic acid and nickel chelate is then used to fish out the required molecule by chelating with the polyhistidine tag. 

In the case of gel permeation or size-exclusion HPLC (HP-SEC), selectivity arises from differential migration of the biomolecules as they permeate by diffusion from the bulk mobile phase to within the pore chambers of the stationary phase. Ideally, the stationary phase in HP-SEC has been so prepared that the surface itself has no chemical interaction with the biosolutes, with the extent of retardation simply mediated by the physical nature of the pores, their connectivity, and their tortuosity. In this regard, HP-SEC contrasts with the other modes of HPLC, where the surfaces of the stationary phase have been deliberately modified by chemical procedures by (usually) low molecular weight compounds to enable selective retardation of the biosolutes by adsorptive processes. Ideally, the surface of an interactive HPLC sorbent enables separation to occur by only one retention process, i.e., the stationary phase functions as a monomodal sorbent. In practice with porous materials, this is rarely achieved with the consequence that most adsorption HPLC sorbents exhibit multimodal characteristics. The retention behavior and selectivity of the chromatographic system will thus depend on the nature and magnitude of the complex interplay of intermolecular forces... 

Solute retention in reversed-phase HPLC is dependent on the different distribution coefficients established between a polar mobile and a nonpolar stationary phase by the peptidic components of a mixture. Although there are many similarities between reversed-phase HPLC separations of peptides and the classical liquid-liquid partition chromatographic methods, it is debatable whether the sorption process in reversed-phase HPLC arises due to partition or adsorption events, i.e., whether the nonpolar stationary phase functions as a bulk liquid or as an adsorptive monolayer. These aspects and the theoretical models for reversed-phase HPLC are discussed in a subsequent section. 

In GSC, separation occurs based on differences in the adsorption of the various components in the sample onto the solid adsorbent. While GSC may not offer as much flexibility in stationary phase functionality as GLC, it has its own advantages. For separation applications, advantages include higher available operating temperatures, higher column efficiencies, and no stationary phase leakage. Typical solid phases for GSC include zeolites, silica gel, activated alumina, carbon, carbon molecular sieves, diatomites, and porous polymers. 

While lome sutionaiy phases are dominated a single nu mecha alsm, and subsequently can be placed near one of the apices of the triangle, other stationary phases function via two or more mqjor retention mecha nisms. These multimodal phases, henceforth referred to as mixed-interaction or mixed-mode stationary phases, have historically bMn successful in separating such biomolecules as nucleic adds and protdns. Most often, phases of these types have been used in low- to medium-pressure chromatography, and have often been of polymeric base. 

Because of the development of analytical methods for determination of MC, especially in last decade, the most widespread analytical techniques for their determination are commercialized enzyme-linked immunosorbent assays and HPLC with diode array detectors of anion-exchange columns for this purpose were only sporadically reported with UV detection and isocratic or gradient ° elution. For anion-exchange stationary phase functionalized with diethylaminoethylene groups, better resolution for LR and YR was reported than for Cl8 columns.For separation of cyanobacterial toxins, including MC-LR and -RR, hydrophilic interaction HPLC with gel Amide-80 column and amide C16 column providing comparable resolution to that of conventional reversed-phase Cl8 columns were applied. 

Method Stationary-phase functional group Eluent Analytes... 

Apolar stationary phases having no dipolar moments, that is their center of gravities of their positive and negative electric charges coincide. With this type of compound, the components elute as a function of their increasing boiiing points. The time difference between the moment of injection and the moment the component leaves the column is called the retention time. 

Nonideal asymmetrical chromatographic bands showing (a) fronting and (b) tailing. Also depicted are the corresponding sorption isotherms showing the relationship between the concentration of solute in the stationary phase as a function of its concentration in the mobile phase. 

A few GLC stationary phases rely on chemical selectivity. The most notable are stationary phases containing chiral functional groups, which can be used for separating enantiomers. ... 

Limitations with the chiral selectivity of the native cyclodextrins fostered the development of various functionalized cyclodextrin-based chiral stationary phases, including acetylated (82,83), sulfated (84), 2-hydroxypropyl (85), 3,5-dimethylphenylcarbamoylated (86) and... 

Although the chiral recognition mechanism of these cyclodexttin-based phases is not entirely understood, thermodynamic and column capacity studies indicate that the analytes may interact with the functionalized cyclodextrins by either associating with the outside or mouth of the cyclodextrin, or by forming a more traditional inclusion complex with the cyclodextrin (122). As in the case of the metal-complex chiral stationary phase, configuration assignment is generally not possible in the absence of pure chiral standards. 

Thermodynamic paths are necessary to evaluate the enthalpy (or internal energy) of the fluid phase and the internal energy of the stationary phase. For gas-phase processes at low and modest pressures, the enthalpy departure function for pressure changes can be ignored and a reference state for each pure component chosen to be ideal gas at temperature and a reference state for the stationarv phase (adsorbent plus adsorbate) chosen to be adsorbate-free solid at. Thus, for the gas phase we have... 

In ion-exchange chromatography (lEC), the mobile phase modulator is typically a salt in aqueous solution, and the stationary phase is an ion-exchanger. For ddnte conditions, the solute retention faclor is commonly found to be a power-law function of the salt uormahty [cf. Eq. (16-27) for ion-exchange equilibrium]. 

TABLE 16-15 Concentrations and h-Function Roots for Displacement Chromatography of a Mixture of M-1 Components Numbered in Order of Decreasing Affinity for the Stationary Phase (Adapted from Frenz and Horvath/ 1985). 

It is seen that equations (13) and (15) are very similar to equation (10) except that the velocity used is the outlet velocity and not the average velocity and that the diffusivity of the solute in the gas phase is taken as that measured at the outlet pressure of the column (atmospheric). It is also seen from equation (14) that the resistance to mass transfer in the stationary phase is now a function of the inlet-outlet pressure ratio (y). 

Thus as (y) will always be greater than unity, the resistance to mass transfer term in the mobile phase will be, at a minimum, about forty times greater than that in the stationary phase. Consequently, the contribution from the resistance to mass transfer in the stationary phase to the overall variance per unit length of the column, relative to that in the mobile phase, can be ignored. It is now possible to obtain a new expression for the optimum particle diameter (dp(opt)) by eliminating the resistance to mass transfer function for the liquid phase from equation (14). 

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