Packing Materials

Porous silica is the most widely used adsorbent in HPLC, although extensive work has been conducted with alumina, zirconia and polymer supports. The majority of these supports are spherical and, for analytical applications with small molecules, have general physical parameters of pore size 10 nm, pore

Figure 9 Stand-alone guard column.

Figure 9 Stand-alone guard column.

volume 0.5 mL g_1 and surface area 250 m2 g_1. The most common particle size used in columns is 5 |m. Care must be taken when using the particle size given by a manufacturer. What is meant by 5 |im particles, is actually a 5 |im particle size distribution. Depending upon the type and mode of measuring instrument used by the manufacturer, there will be a particle size distribution based on number, area or volume.

In the number distribution the percentage of the particles in each size range is determined by dividing the number in each range by the total number of particles. In the area distribution, the percentage of the surface area in each size range is determined by dividing the surface area calculated for each size range by the total surface area of all the particles. This has the effect of emphasizing the larger particles more than the number distribution. For example, one particle of 10 |im diameter has the same surface area as 100 particles of 1 |im diameter. The volume distribution is calculated from the percentage of the volume of each particle size divided by the total volume of all the sizes. The volume distribution emphasizes the large particles even more than the area distribution, e.g. a 10 |m diameter particle has the same volume as 1000 particles of 1 |m. Typical distributions (for a 5 |m Spherisorb) are given in Figure 10, showing the emphasis on the larger particle sizes in the distributions for area and volume.

Silica particles used for packing are commonly manufactured from a sol-gel type process, starting from a cation-based sol. This produces gels that are high in their cationic species, for example Spherisorb has a sodium content of 1500 ppm. Newer manufacturing methods introduced in recent years, use organic tetraethyl orthosilicate (TEOS) condensation systems that can produce exceptionally pure silicas.

In the sol-gel process, the size of the particles can be controlled by the viscosity of the mixture, the emulsification rate and the amount and type of surfactant used to stabilize the emulsion. However, none of the processes can produce monodispersed particles - they produce a particle size distribution and the required particle size is then obtained from this by air classification or elutriation.

The tighter the particle size distribution, the more uniform a column packing can be made. However, with very tight particle size distributions it becomes more difficult to pack the columns. Van Deemter curves demonstrating the effect of particle size on the column efficiency are shown in Figure 11.

The surface area, pore size and pore volume of packing material are determined either by nitrogen BET or mercury porosimetry. As with particle size distributions, care must be used in the interpretation of these data because different models are used by

Figure 10 Particle size distributions for a 5 |m Spherisorb. (A) Number; (B) area; (C) volume.

different manufacturers, which can impose different interpretations on the type and shape of pores. In HPLC, an important factor is the absence of micropores and mesopores. Micropores are defined as having a diameter of less than 1 nm, while mesopores are less than 5 nm in diameter. If these pores are included

Flowrate I Figure 11 Van Deemter curves.

in the surface area calculations, then very large surface area values are obtained. However, most analyte molecules cannot penetrate into micropores and even if they could possibly penetrate into the mesopores, diffusion is very restricted by steric hindrance, which in turn affects mass transfer and hence efficiency.

Silica is a preferred adsorbent in HPLC as the surface characteristics can be modified by chemical reaction to change the hydrophilic silica surface into a hydrophobic surface suitable for reversed-phase chromatography, or to place an ionic exchange material on the surface for ion exchange chromatography.

A very broad range of functionality can be attached to the silica surface, through reaction with or-ganosilanes, the most popular being C18 or octadecyl-silane. The linkage onto the silica is of the type Si-O-Si-R, where R is the functional group. Manufacturers use a variety of silanes, ranging from mono-chloro to trichlorosilanes to mono and tri methoxy and ethoxy silanes. Attachment of the monofunc-tional silane onto the surface of the silica leads to a monomeric phase, while the addition of small amounts of water into the bonding process can lead to polymeric phases.

Manufacturers normally give bonding figures as % carbon loadings. This is a very misleading figure as the percentage of carbon loading alone is not a relevant parameter because of differences in the surface area of the original silica, which result in different surface densities of the bonded alkyl groups. Table 3

Table 3 Column packing parameters

Description

Column volume

Si density

Mass ofpacking

Carbon load

Amountofcarbon in the

(mL)

(gmL ~1 )

(g)

(%)

column (g)

Nova-Pak C18

1.8

0.91

.64

7

0. 2

iBondapak C18

1.8

0.46

0.82

0

0.08

Table 4 Some commercially available bonded phases

Phase name

Usage

Comments

C1 Purification

C4 Protein separations

C6 Protein separations

C8 General purpose

C18 ODS General purpose

Phenyl Separation of complex mixtures

Cyano CN Broad spectrum of mixtures with different polarities

Amino NH2 Sugar analysis and for aromatic compounds

Diol Complex mixtures

Ion exchange Ionic species

Chiral phases Enantiomer separations

Almost 80% of all applications have been developed with C8 and C18 phases

Almost 80% of all applications have been developed with C8 and C18 phases

Induced dipole interactions with polar analytes Can be used in both normal and reversed phase

Weak anion exchanger

Slightly polar adsorbent for normal phase

Four major types available: donor, polymer, cavity and exchange

ODS, octadecylsilica.

outlines this problem, showing the amount of carbon on a material against its percentage carbon loading.

Table 4 provides a broad summary of the bonded phases that are commercially available. It must be stressed that this is in no way an extensive list. There are many specialized phase-column combinations supplied by many manufacturers. In particular, these speciality phases tend to be in the chiral separation areas.

The term end-capping was introduced in the late 1970s to indicate that a secondary treatment had been applied to a bonded phase. This was an attempt to cover all available silanols. The most common end-capping agent is trimethylchlorosilane; this molecule is relatively small and so can penetrate into the pore system of the adsorbent. Generally end-capping leads to an improvement in the chromatography of basic compounds. However, even after the best end-capping, it is estimated that about 50% of all silanols are still unbonded.

A term that has recently been introduced into column packings is 'base-deactivated' reversed phases. This is a particularly unfortunate term since these packings are not deactivated with bases but have surface treatments or treatments of the silica that make them particularly useful for separating basic compounds. Amines, for example, tend to tail on conventional reversed-phase column packings because of the residual acidic silanols. These base-deactivated packings are normally manufactured from the new pure silicas and have a more uniform or reduced level of silanols.

A major problem with silica is the small pH range over which the adsorbent can be used. Above pH 8.5 the siloxane bridge is broken, leading to breakdown through solvation of the particle. Below pH 2.5, the bonded phase is hydrolysed from the surface of the silica. In an attempt to extend the pH range of silica, attempts have been made to manufacture pH-stable bondings. These have normally been based on coating the silica with a polymer, such as polybutadiene or a grafted polysiloxane.

Other ceramics have been reported as packing materials. The most common are alumina, zirconia and titania. Although it is possible to attach hydrophobic groups onto all of these materials, with alumina the silane is very easily removed from the surface. To obtain a hydrophobic character for alumina, poly-butadiene or grafted polysiloxane polymers are used. These types of bondings have been divided into two groups. The first comprises those polymers with siloxane bonds such as polymethyloctylsiloxane, polymethyloctadecylsiloxane and silica monomers, while the second is represented by purely organic monomers and oligomers such as polybutadiene.

Another support that has been developed and can be used under very aggressive conditions is porous graphitic carbon.

Polymer-based adsorbents are also used as a support material in HPLC columns. They are manufactured from synthetic cross-linked organic polymers. Their main application area is in size-exclusion chromatography and ion exchange chromatography. In normal-phase and reversed-phase HPLC they still have very few applications. A problem is that even though the new polymer packings are highly cross-linked and stable, they still carry the history of their precursors and they can swell with some solvents.

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