Sorbents, HPLC

Having wide and increasing quantity of RP HPLC sorbents in disposal the main question in RP HPLC is their interchangeability. Column chai acteristics that ai e usually described by their manufacturers are not full enough for the analytic to choose a suitable column for the specified resolutions or he ought to choose other similar column used before. In fact, nomenclature of reversed-phase stationai y phases is too unsophisticated and is a source of confusion in their application. 

Recently, new approaches of sorbent construction for reversed-phase chromatography have been developed. Silicas modified with hydrocarbon chains have been investigated the most and broadly utilized for these aims. Silica-based materials possess sufficient stability only in the pH 2-8 range. Polymeric HPLC sorbents remove these limitations. Tweeten et al. [108] demonstrated the application of stroongly crosslinked styrene-divinylbenzene resins for reversed-phase chromatography of peptides. 

With bonded, NP-HPLC sorbents, such as the porous aminopropylsilica sorbents, the distribution constant, abSii, can be equated with the solubility parameter, 6, which in turn is a measure of the intermolecular interaction energy per unit volume of the polypeptide in a pure liquid such that... 

Silica particles used for SPE sorbents are typically irregularly shaped, 40 to 60 pm in diameter. Silica particles used for sorbents in high-performance liquid chromatographic (HPLC) columns are generally spherical and 3 to 5 pm in diameter. Due to the differences in size and shape, SPE sorbents are less expensive than HPLC sorbents. Much greater pressures are required to pump solvents through the smaller particle sizes used in HPLC. 

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... 

As noted, the retention of a polypeptide or protein with HP-IEX sorbents primarily arises from electrostatic interactions between the ionized surface of the polypeptide or protein and the charged surface of the HPLC sorbent. Various theoretical models based on empirical relationships or thermodynamic considerations have been used to describe polypeptide and protein retention, and the involvement of the different ions, in HP-IEC under isocratic and gradient elution conditions (cf. Refs.6,19 33 40,78-90). Over a limited range of ionic strength conditions, the following empirical dependencies derived from the stoichiometric retention model can be used to describe the isocratic and gradient elution relationships between the capacity factor In and the corresponding salt concentration [C,] or the median capacity factor In k ex, and the median salt concentration [C,] of a polypeptide or protein solute, namely,... 

The excellent reproducibility that can be achieved in repetitive separations carried out over long periods of time, due in part to the stability of the various stationary phases to many aqueous mobile phase conditions. Thus, it is not uncommon with the current generation of pressure-stable HPLC sorbents for little change in the resolution to arise after more than 1000 repetitive analytical separations. 

FIGURE 16 Schematic representation of the origins of zone-broadening behavior and mass transfer effects of a polypeptide or protein due to Brownian motion, eddy diffusion, mobile phase mass transfer, stagnant fluid mass transfer, and stationary-phase interaction transfer as the polypeptide or protein migrated through a column packed with porous particles of an interactive HPLC sorbent. 

The consequences of the type of thermodynamic behavior manifested by the polypeptides 1 and 2, and other polypeptides and proteins in their interactions with the different classes of ligates found in HPLC sorbents are... 

All of these effects impact on the loading capacity of a particular HPLC sorbent, which can thus exhibit subtly different selectivity-capacity dependencies with different classes of polypeptides and proteins. Such behavior has been documented8,78,160,150,227-230 for enzymes and other proteins in a variety of studies. For example, when conformational reordering of a protein structure occurs in both the mobile phase and the stationary phase, this will... 

Other phenomena besides conformational processes can also lead to multizoning effects with polypeptides and proteins when they interact with adsorptive HPLC sorbents. The so-called split peak effect is probably the easiest of these phenomena to be identified and steps taken to remedy. The split peak effect is very often seen in HP-BAC, RP-HPLC, and HP-HIC and to a lesser extent in the HP-IEX of proteins.325-327 This effect is manifested by the presence of a weakly retained (or occasionally as a nonretained peak) and a more strongly retained peak with the bound-to-free ratio between the weakly retained to strongly retained species dependent on the diffusion and adsorption kinetics. An extreme case of the split peak effect involves the weakly interacting component elution in or near to the column breakthrough volume. In this case, the amount of protein in the breakthrough zone is influenced by the nominal pore diameter and ligand density of the sorbent, the flow rate, and the injection volume. This effect can be circumvented by... 

HI TABLE 10 General Form of the Commonly Employed Isothermal Expressions Used for the Analysis of the Binding Behavior of Polypeptides and Proteins with HPLC Sorbents... 

At this stage of developments, most fixed-bed adsorption models assume that film mass transfer resistance is small compared with the other transport resistances in the system and that equilibrium is reached instantaneously between the solute in the pore liquid and at the surface of the sorbent. Even if it assumed that a homogeneous adsorptive HPLC sorbent is used, it can be readily shown, however, that both film and pore diffusion mass transfer resistances cannot be ignored393,394 and that the dynamic behavior of the adsorption stage is greatly dependent on the rate of the polypeptide- or protein-ligate interaction.8,357,395 Breakthrough of solute(s) may thus occur... 

A useful literature relating to polypeptide and protein adsorption kinetics and equilibrium behavior in finite bath systems for both affinity and ion-ex-change HPLC sorbents is now available160,169,171-174,228,234 319 323 402"405 and various mathematical models have been developed, incorporating data on the adsorption behavior of proteins in a finite bath.8,160 167-169 171-174 400 403-405 406 One such model, the so-called combined-batch adsorption model (BAMcomb), initially developed for nonporous particles, takes into account the dynamic adsorption behavior of polypeptides and proteins in a finite bath. Due to the absence of pore diffusion, analytical solutions for nonporous HPLC sorbents can be readily developed using this model and its two simplified cases, and the effects of both surface interaction and film mass transfer can be independently addressed. Based on this knowledge, extension of the BAMcomb approach to porous sorbents in bath systems, and subsequently to packed-, expanded-, and fluidized-bed systems, can then be achieved. 

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