Pore size, stationary-phase particles

Two classes of micron-sized stationary phases have been encountered in this section silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 50 to 4000 A for silica particles and from 50 to 1,000,000 A for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography, also called molecular-exclusion or gel-permeation chromatography, separation is based on the solute s ability to enter into the pores of the column packing. Smaller solutes spend proportionally more time within the pores and, consequently, take longer to elute from the column. 

In exclusion chromatography, the total volume of mobile phase in the column is the sum of the volume external to the stationary phase particles (the void volume, V0) and the volume within the pores of the particles (the interstitial volume, Vj). Large molecules that are excluded from the pores must have a retention volume VQ, small molecules that can completely permeate the porous network will have a retention volume of (Vo + Fj). Molecules of intermediate size that can enter some, but not all of the pore space will have a retention volume between VQ and (V0 + Fj). Provided that exclusion is the only separation mechanism (ie no adsorption, partition or ion-exchange), the entire sample must elute between these two volume limits. 

Size exclusion chromatography. The separation occurs because the stationary phase particles are porous and the small molecules enter the pores and are slowed from passing through the column, while the large molecules pass through more quickly since they do not enter the pores. 

Porous microparticles are the most common stationary phase particles used in modern HPLC. The role of pore size is a critical one, as the pores provide the surface with which the sample interacts. Particles with small pores exhibit a high surface area and therefore have greater retention. Large molecules like proteins, however, may be excluded from the small pores, and for those molecules a packing with a larger pore size is preferable. The difference between porous particles, pellicular particles, and porous microparticles is illustrated in Figure 3.19. Porous particles are seldom used owing to low efficiencies and are not discussed further. 

So Get the stationary phase particle size as small as possible, get the pore size optimized and you re sailing. Slow down - it s not quite so simple. There are other major effects that have to be considered when scaling a preparative separation. The friction caused by the eluent passing over stationary phase particles generates heat, which in turn reduces the viscosity of the solvent. Cooling by conduction in the vicinity of the column walls reduces the viscosity of the solvent close to the wall in comparison to that at the centre of the column. Consequently, the solvent at the centre of the column is now travelling at a... 

The third and fourth terms of Eq. (3) also relate to mass transfer. The third term, Cjx, describes the contribution of mass transfer of solutes to and from areas in the column where the mobile phase is stagnant (e.g., within the pores of the packing). The size of this term is related to stationary-phase particle diameter and solute diffusion coefficient according to... 

Resolution of the protein separation is affected by several stationary phase properties. Reduction of the particle diameter is obviously the most straightforward way to improve the efficiency. For analytical purposes, 3- to 10-pm particles are a good compromise between chromatographic performance and the required pressure. The pore size of the particles should be a minimum of 300A to provide accessibility for large proteins. 

Figure 1 Retention mechanisms in LC (A) partition (B) adsorption (C) ion-exchange and (D) size-exclusion. 1, liquid mobile phase 2, sample molecules or ions, shown as circles (the latter and 2 are connected by lines) 3, stationary phase (one particle in case (A), 3 is a support particle covered with thin film of liquid, 4 being the stationary phase itself) 5, an inner pore in a stationary phase particle 3.

Based on the above-mentioned six key properties of reversed phases, the stationary phases can be characterized. A wide variety of literature exists on this subject [9-15]. Of course, the synthesized stationary phases can be subjected to a full physicochemical examination (nitrogen adsorption measurements to determine the specific surface area, the pore volume and the pore size, CHN analysis to determine the surface coverage of the stationary phase, particle size measurements, etc.). However, all these characterizations are not really to the point, because in the end only the chromatographic separation counts. As a result, chromatographic tests for the characterization and classification of reversed phases have established themselves, from which a representative few, without any claim to being exhaustive, are presented here (Figures 4.1—4.3). 

Polymer Molar masses Critical range Used samples Mobile phase Solvent / Nonsolvent Adsorb / Desorb Stationary phase Particle diameter Pore size Column dimension Conditions Temperature Flow rate Inj. volume Detector Analytical application and / or notices Investigators Reference... 

In real-life situations, particles in solution do not have a fixed size, resulting in the probability that a particle that would otherwise be hampered by a pore passing right by it. Also, the stationary-phase particles are not ideally defined both particles and pores may vary in size. Elution curves, therefore, resemble Gaussian distributions. The stationary phase may also interact in undesirable ways with a particle and influence retention times, though great care is taken by column manufacturers to use stationary phases that are inert and minimize this issue. 

Preparation of the stationary phase was performed by dissolving 0.75 g of amylose tnXcyclohexyl carbamate) in 10 ml of tetrahydrofuran. Macroporous silanized silica gel (3 g) [made from silica gel, Daiso Gel SP-1000, pore size 100 nm, particle size 7 /u-m, silanized using (3-aminopropyl)triethoxysilane in benzene at 80°C] was added to the solution and the resulting suspension was dried under vacuum. A portion of this material (1.5 g) and fluorescent indicator (Merck) (0.1 g) were mixed with methanol (3 ml). The slurry was applied to microslides (2.6 X 7.6 cm, thickness 0.3 mm) and the plates were dried in an oven at 110°C for 30 min [37]. 

In the development of a SE-HPLC method the variables that may be manipulated and optimized are the column (matrix type, particle and pore size, and physical dimension), buffer system (type and ionic strength), pH, and solubility additives (e.g., organic solvents, detergents). Once a column and mobile phase system have been selected the system parameters of protein load (amount of material and volume) and flow rate should also be optimized. A beneficial approach to the development of a SE-HPLC method is to optimize the multiple variables by the use of statistical experimental design. Also, information about the physical and chemical properties such as pH or ionic strength, solubility, and especially conditions that promote aggregation can be applied to the development of a SE-HPLC assay. Typical problems encountered during the development of a SE-HPLC assay are protein insolubility and column stationary phase... 

The two techniques differ in that HDC employs a nonporous stationary phase. Separation is affected as a result of particles of different size sampling different velocities in the interstitial spaces. Size exclusion chromatography is accomplished by superimposing a steric selection mechanism which results from the use of a porous bed. The pore sizes may vary over a wide range and the separation occurs as a result of essentially the same processes present in the gel permeation chromatography of macromolecules. 

If a single pore size is employed in the stationary phase, which is larger than the largest particle to be analyzed, the technique has been termed porous HDC. A model for the separation in this type of system has been described by DiMarzio and Guttman (12, 13). 

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