Porous layer beads

Finally, the useful life of an analytical column is increased by introducing a guard column. This is a short column which is placed between the injector and the HPLC column to protect the latter from damage or loss of efficiency caused by particulate matter or strongly adsorbed substances in samples or solvents. It may also be used to saturate the eluting solvent with soluble stationary phase [see Section 8.2(2)]. Guard columns may be packed with microparticulate stationary phases or with porous-layer beads the latter are cheaper and easier to pack than the microparticulates, but have lower capacities and therefore require changing more frequently. 

Figure 1. A, porous particle used to illustrate slow mass trarlsfer due to diffusion in the stag-nant mobile phase within the particle. B, illustration of a porous layer bead.

A wide variety of bases, nucleosides and nucleotides have been separated using porous layer bead ion exchangers. A representative chromatogram of the separation of ribonucleoside mono-phosphoric acids from the work of Smukler ( ) is shown in Figure 4. Recently, ion exchangers chemically bonded to small particle diameter (> 10 ym) silica have been successfully applied to the separation of nucleic acid constitutents (37). The rapid separations using such supports undoubtedly mean that they will find increasing use in the future. 

Figure 6, High pressure liquid chromatogram of creatine kinase isoenzymes. First peak, MM second peak, BB. Conditions 50 cm X 4.8 mm (i.d.) column with yydac porous layer bead anion exchange mobile phase, step gradient Solvent A, 10 mmol/liter Tris buffer, pH 8.3 solvent B, 10 mmol/liter Tris buffer, pH 7.0,0.5 mol KCl flow rate, 2 ml/min detection, collected fractions assayed (45).

The iiqwrteuice of porous layer beads in modem liquid Chromatography declined when totally porous silica mlcroparticlai3 with diameters less than 10 micrometers, and with narrow particls size ranges, became available (Figure 4.1) [4]. Indeed, vba... 

Property Porous Layer Beads Totally Porous Microparticles... 

For use with 25 cm x 4.6 mm analytical columns, guard columns and scavenger columns are often 4.6 mm internal diameter and 3-10 cm in length. They can be packed with microparticulate stationary phases or with porous layer beads. Porous layer beads are cheaper than microparticulates and are easier to pack, but they have lower capacities and will require changing more often. It is usually difficult to know how long a pre-column will last before it requires changing. In routine work, precolumns are usually repacked or replaced to a fixed schedule. 

Additionally, they have been used as porous layer beads (PLB) (10, i I), in which case a thin porous adsorbent layer is coated onto a fluid impervi-(Mi> core, snch ns solid glass bends. The thickness of (he porous Inyci is generally 1-3 pm, i.e., to of the particle diameter, and the par-... 

Plate number, 3,4,7, 27, 52, S3 Plates per second, 30-33 PLB, see Porous layer beads Polar group selectivity, 181-183 Polar solvent, selective uptake from eluent by polar adsorbent, 8S Polarizability, 206... 

The solute molecules can enter and leave the particles only by diffusion. The particles are porous, like sponges, and most of the absorbent surface area is given by the surface of the inner pores. In bLC most of the stationary liquid phase is also inside the particles. Ofily porous layer beads which have been used in the past and may find sonie applications in the future represent an exception. The diffusion through the particles takes some time. The average time necessary for a molecule to diffuse across a distance dp is... 

The most commonly used column support materials are made from diatomite. Other materials include sand. Teflon, inorganic salts, glass beads, porous layer beads, porous polymers, carbon blacks, etc. We will discuss the diatomite supports in some detail and additional information may be obtained in Chapter 3. 

The adsorbents in HPLC are typically small-diameter, porous materials. Two types of stationary phases are available. Porous layer beads (Figure 3.14A) have an inert solid core with a thin porous outer shell of silica, alumina, or ion-exchange resin. The average diameter of the beads ranges from 20 to 45 fim. They are especially useful for analytical applications, but, because of their short pores, their capacities are too low for preparative applications. 

Microporous particles are available in two sizes 20 to 40 jam diameter with longer pores and 5 to 10 fim with short pores (see Figure 3.14B). These are now more widely used than the porous layer beads because they offer greater resolution and faster separations with lower pressures. The micro-porous beads are prepared from alumina, silica, ion-exchanger resins, and chemically bonded phases (see next section). 

HPLC in the adsorption mode can be carried out with silica or alumina porous-layer-bead columns. Small glass beads are often used for the inert core. Some of the more widely used packings are /a Porasil (Waters Associates), BioSilA (Bio-Rad Laboratories), LiChrosorb Si-100 Partisil, Vydac, ALOX 60D (several suppliers), and Supelcosil (Supelco). 

The retention factor is directly proportional to the volume occupied by the stationary phase and more especially to its specific area (m g ) in the case of adsorbents. A column packed with porous-layer beads produces lower k values and hence shorter analysis times than a column containing completely porous particles if the other conditions remain constant. Silica with narrow pores produces larger k values than a wide-pore material. 

Various types of stationary phases are in use Porous particles, nonporous particles of small diameter, porous layer beads, perfusive particles, and monolithic materials. 

These are large particles with a diameter in the 30 gm range which allows to pack them dry. They consist of a nonporous core (e.g. from glass) which is covered with a 1 -3 gm layer of a chromatographically active material. Porous layer beads (PLBs) are rarely used nowadays but can be found in guard columns or as repair material for deteriorated columns with collapsed packing. 

The column may also be opened to inspect the chromatographic bed. If the packing has collapsed (Figure 7.18a), then the column is repaired in the following way (b) the excess packing is removed with a fine spatula and the bed smoothed over (c) dead volumes are filled with glass beads of about 35 pm or suitable porous layer beads and the column is resealed. The frit is replaced by a new one. ... 

Microporous particles (3-10 (im) give columns that are as much as 20 times as efficient as porous layer-bead or pellicular (40 pm) packings. Whilst modem LC is based almost exclusively on microporous packing materials it is informative to relate the advances in particle design with the attempts to eliminate the deleterious effects on column performance since the latter as expressed by H is related to experimental variables, such as, the particle size (dp), the nominal stationary phase thickness (ds) and the mobile phase velocity (u). 

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