Bonded phases stability

The final part of the chapter provides a refresher on pA from an analytical chemist s perspective, the drivers for choosing normal phase versus reversed phase as a separation mode for a particular analysis, and instru-ment/system consideration, and it concludes with a very interesting section on column testing within the framework of bonded phase stability (effect of pH and type of buffer) and probing column selectivity. 

Choice of Buffer Related to Bonded-Phase Stability... 

Different types of buffers at the same ionic strength and wpH can have a significant impact on the dissolution of silica. The dissolution of silica is usually measured by the silicomolybdate colorimetric method [41]. When determining the bonded-phase stability using different run buffers (effect of buffer counteranion or countercation), the same H must be used. The H values (pH of the mobile phase aqueous -i- organic) may be different from the aqueous portion of the mobile phase and may obscure if the dissolution of the silica is directly related to the type of anion/cation and/or the pH. Generally, with the addition of organic solvents the pH of the mobile phase decreases for basic buffers and increases for acidic buffers (see Section 4.5 for more details). 

For point 2, column performance can be adversely affected by pumping multiple volumes of mobile phase through the column due to (a) changes in the bonded-phase stability at various temperatures, pH values, and pressures and (b) accumulation of retained analyte components on the stationary phase. The maximum pH, temperature, and backpressure for which the column can operate versus the maximum number of column volumes should be known in order to avoid discrepancy in the data generated between laboratories (new versus aged column). For method transfer it is suggested that brand new unused columns (manufactured less than one year) should be used. 

An important property of these siloxane phases is their stability under the conditions used in most chromatographic separations the siloxane bonds are attacked only in very acidic (pH < 2) or basic (pH > 9) conditions. A large number of commercial bonded-phase packings are available in particle sizes suitable for HPLC.48... 

The drawback of the described adsorbents is the leakage of the bonded phase that may occur after the change of eluent or temperature of operation when the equilibrium of the polymer adsorption is disturbed. In order to prepare a more stable support Dulout et al. [31] introduced the treatment of porous silica with PEO, poly-lV-vinylpyrrolidone or polyvinylalcohol solution followed by a second treatment with an aqueous solution of a protein whose molecular weight was lower than that of the proteins to be separated. Possibly, displacement of the weakly adsorbed coils by the stronger interacting proteins produce an additional shrouding of the polymer-coated supports. After the weakly adsorbed portion was replaced, the stability of the mixed adsorption layer was higher. 

The polymeric resin beads fill a need that arises from the instability of silica gel and its products to mobile phases of extreme pH (outside a pH range of about 4.0-7.0) and, consequently, are employed in most ion exchange separations. Organic moieties containing ionic groups can be bonded to silica and produce an effective ion exchange media, but the restrictions of pH on phase stability still apply. It follows that ion exchange bonded phases are less popular than the polymer bead alternatives. 

It is shown that the stabilities of solids can be related to Parr s physical hardness parameter for solids, and that this is proportional to Pearson s chemical hardness parameter for molecules. For sp-bonded metals, the bulk moduli correlate with the chemical hardness density (CffD), and for covalently bonded crystals, the octahedral shear moduli correlate with CHD. By analogy with molecules, the chemical hardness is related to the gap in the spectrum of bonding energies. This is verified for the Group IV elements and the isoelec-tronic III-V compounds. Since polarization requires excitation of the valence electrons, polarizability is related to band-gaps, and thence to chemical hardness and elastic moduli. Another measure of stability is indentation hardness, and it is shown that this correlates linearly with reciprocal polarizability. Finally, it is shown that theoretical values of critical transformation pressures correlate linearly with indentation hardness numbers, so the latter are a good measure of phase stability. 

These separations can be carried out using a silica-based bonded phase however, the important advantage of organic polymer stationary phase materials is their chemical stability. The columns can be washed by using an alkaline solution after a certain number of injections. According to the chromatograms, the proteins in serum are completely eluted and nothing remains inside the column. However, the pressure drop in this type of analysis... 

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