Packing Stationary Phase

A very important factor for successful HPLC separation is the correct choice of column packing. In the past, many different packing materials were tested, for instance, powdered sugar, diatomaceous earth, aluminum oxide, calcium carbonate, calcium hydroxide, magnesium oxide. Fuller s earth, and silica. Today, silica is generally u.sed as the basis for adsorbent chromatography aluminum oxide is also used to a certain extent. Both materials are pressure stable and are unaffected by pH over a wide range. 

Particle Size, Particle-Size Distribution. Standard particle size for analytical HPLC columns is 5 or 10 pm. The smallest commercially available particle size is 1.5 pm. The particle-size distribution should be as naiTOw as possible so that good packings and therefore good separations are obtainable. The smaller the particle-size distribution of the stationary phase, the higher is its quality and therefore its price. However, if the size of stationary phase particles is absolutely identical, this can cause problems in packing the column. 

Pore Diameter, SpeciHc Surface Area. In adsorption chromatography a packing material with a very high specific surface area is required. The larger the surface area, the more active sites are available for (adsorptive) interaction. In the case of spherical particles (10-pm i.d.) I % of the geometrical surface contributes to the total surface area. The dominant remainder of the surface area originates from the pores of the particles. A normal silica phase has a specific surface area of ca. 3(X)m /g, a mean pore diameter of lOnm. and a mean pore volume of ca. 0.5- 1.0 mL/g. 

Most analytes have no access to pores with a diameter smaller than 3-4 nm. Therefore the packing material should show a narrow pore-size distribution so that the total number of very small pores (to which the analytes have no access) is as low as possible. Generally, the pore diameter should be about 3-4 times larger than the diameter of the analyte in solution, to ensure sufficient accessibility. 

Nonporous packings with very small particle diameters have developed into useful tools for fast and economic separations. Due to the reduced resistance to mass transfer of the nonporous material, separation efficiency is increased at high flow 

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A large variety of unique column packings (stationary phases) used in HPLC provide a wide range of selectivity. 

The pump that is used in HPLC cannot be just any pump. It must be a special pump that is capable of very high pressure (up to 5000 psi) in order to pump the mobile phase through the tightly packed stationary phase at a reasonable flow rate, usually between 0.5 and 4.0 mL/min. It also must be nearly free of pulsations so that the flow rate remains even and constant throughout. Only manufacturers of HPLC equipment manufacture such pumps. 

Column packing (stationary phase) suspended in solvent (mobile phase)... 

Figure 2.12 Reversed-phase HPLC of a sample composed of two compounds, one polar, the other nonpolar. The column packing (stationary phase) is symbolized by spheres and labeled Oil and the mobile phase as wavy lines labeled Water. The polar molecules are shown remaining in the mobile phase (water), while the nonpolar molecules enter the stationary (oil) phase. Finally, the chromatographic profile illustrates that in this case the polar molecule will not be retained and will emerge with a shorter retention time than the nonpolar molecule.

Figure 14.1. Flow profile for injected solute travelling through a packed stationary phase.

Column packing stationary phase material in the form of small solid particles which may be coated with a liquid stationary phase. Particle size is 3-20 pm in HPLC, 50 500 pm (60-100 mesh) in GC. 

As first observed in 1903 by M. Tswett, a Russian botanist, when plant pigments were dissolved in a nonpolar solvent such as hexane and this solution was passed through a glass column packed with calcium carbonate, a separation of the two major forms of chlorophyll occurred due to a differential migration through the packed stationary phase. This observation of color writing led to the most used term in the separation sciences today—chromatography ... 

Mix 2 g of polymer with 2mL of methanol-water (1 +lv/v) in a test tube and sonicate until complete homogeneity of the mixture is reached. Transfer the suspension rapidly into an empty 100 x 3.9 mm HPLC column mounted on-line with a second empty column to serve as a column packaging device (Fig. 14). Close the column and run the HPLC in pressure-constant mode with methanol-water (1 +1 v/v) as a mobile phase, gradually increasing the pressure from 1 to 25 MPa by properly increasing the flow rate. Flush the system for about 20 column volumes. Disassemble the packing device carefully by cutting the packed stationary phase at the outlet of the double female joint with a spatula or a knife (note 7). 

Although successful operation of microfabricated GC systems has clearly been demonstrated, performance variability has been poor when compared to cmiventional GC technology. This is often associated with the ability to deposit or pack stationary phase materials in a homogeneous fashion, especially in the rectangular cross-sectional geometries of typical microfabricated channels. 

The most essential component in any liquid chromatograph is the column and the chromatographic packing contained therein. This is where the sample is separated into its individual components. The solute molecules are in an equilibrium between the column packing (stationary phase) and the eluent (mobile phase), and it is this equilibrium which governs the separation. In some types of chromatography this equilibrium involves an interaction between the solute and the column packing, but this is not the case in true SEC, as will be seen later. 

Retention in chromatography is controlled by thermodynamic equilibria. The partition ofthe analyte between the mobile and the stationary phase is in control of the retention factor. This partition can be described by the laws of reversible thermodynamics. Therefore, we also borrow the thermodynamic description of the temperature dependence of equilibria. This is the so-called van t Hoff equation, which is the quantitative expression of the Le Chatelier principle. According to this, the temperature dependence of the retention factor k can be described by 2.9, with R being the general gas constant, AH° the molar enthalpy (heat tone) related to the transition of the analyte from mobile to stationary phase, AS° the molar entropy change for this transition, andj( the so-called phase ratio of the packed stationary phase in the column. 

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