Bonded phase packings

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... 

Residual silanol groups in chemically bonded phases have been associated with a number of undesirable interactions with polar solutes such as excessive peak tailing, irreproducible retention times, and excessively long retention times. These problems are particularly prevalent for amines and other strong bases. A large number of test systems have been proposed to characterize the concentration of residual silanol groups on bonded phase packings, and some representative examples are... 

Besides the above differentiation, restricted-access media can be further subdivided on the basis of the topochemistry of the bonded phase. Packings with a uniform surface topochemistry show a homogenous ligand coverage, whereas packings with a dual topochemistry show a different chemical modification of the pore internal surface and the particle external surface (114). Restricted-access media of the former type are divided into mixed-mode and mixed-function phases, bonded-micellar phases, biomatrix, binary-layered phases, shielded hydrophobic phases, and polymer-coated mixed-function phases. Restricted-access media of the latter type include the Pinkerton s internal surface reversed-phase, Haginaka s internal surface reversed-phase diol, alkyl-diol silica, Kimata s restricted-access media, dual-zone phase, tris-modified Styrosorb, Svec s restricted-access media, diphil sorbents, Ultrabiosep phases. Bio Trap phases, and semipermeable surface phases. 

Separation by composition has been performed not only on silica but also on nonpolar carbonaceous materials and on bonded-phase packings of moderate polarity. The reversed-phase separation of biopolymers, especially of proteins, has gained great importance and is well-known to chromatographers and life scientists. What else should protein mapping be if not separation by composition An example,5) of this important technique is given in Fig. 5. 

Of the polar bonded-phase packings that have been investigated, the interactions between carotenes and nitrile stationary phases are very weak thus the limitations described for silica apply (164). Amino-bonded phases eluted with iso-octane containing 0.5% stabilized tetrahydrofu-ran separate a- and /3-carotene (unresolved) from y-carotcnc canthaxanthin and /3-cryptoxanthin are not eluted (161). The cis isomers of /3-carotene are separated from the all-trans isomer thus amino columns offer an alternative option to alumina columns for determining all-trans-/3-carotene without its cis isomers interfering. 

Reversed-phase separation [6] of polar and non-polar PTH-amino acids may be accomplished using Corasil-Cig bonded phase packing (particle diameter, 37-50 jum) and eluting with water-acetonitrile-isopropanol (100 1.5 1). An example of the reversed-phase separation of sixteen PTH-amino acids is given in Fig.4.4. The limit of detection of... 

End-capping—After silylation, reaction of bonded-phase packing with a reactive small molecule to tie up unreacted silanols on the silica surface. Sharpens peaks from basic compounds. 

Silylation—The first step in forming bonded-phase packings from dried silica and chlorodialkylchlorosilanes. 

Workers at Perkin-Elmer [34] have studied the high-speed separation of PAHs using Cis-bonded phase packings (5pm particles) using both isocratic and gradient elution. The analysis of several PAH standards was performed using the 5 pm bonded phase column with gradient elution... 

Fig. 4.4 Analysis of polyaromatic hydrocarbons. Column two 100x4.6mm id in series, Ci8 bonded-phase packing 3 pm particles. Mobile phase acetonitrile-water, linear gradient from 65 to 90% in 20min, at 1.8mL min-1 inlet pressure 4500psig (31.0mPa) initial, 2700psig (18.6MPa) final ambient temperature UV detector at 254nm. Peaks 1, naphthalene 2, fluorene 3, acenaphthalene 4, phenanthrene 5,...

A final important reproducibility specification should be considered which applies specifically to bonded-phase packings. First, the bonded-phase should be specified as being polymeric or monomeric. If polymeric,information on the % organic or % carbon for the packing and the chemical structure of the bonded phase should be provided. However, as shown before, this information is often not sufficient to determine lot-to-lot chromatographic reproducibility. If the bonded phase is monomeric, data on the % organic or % carbon and chemical structure are also useful, but in addition, the surface coverage calculated from these values (6) should also be provided (EQ. 4). 

Ion suppression. A buffer is added to the mobile phase to adjust the pH so that sample components will be present in their nonionized forms. In this way, ionic sample components can be separated by a reverse-phase mode on a bonded-phase packing, a much easier technique than traditional ion, exchange. 

Reverse phase. A form of chromatography where the packing material surface is relatively nonpolar, and the solvent relatively polar. In a reversed-phase separation, the most polar compounds elute first. This order of elution reverses that found with older, normal-phase separations hence reverse phase. See also Bonded-phase packings. 

Bonded phases may be used in both normal and reverse phase chromatography. When normal phase chromatography is done on bonded phase packings, the packing is more polar than the mobile phase. Polar bonded phases such as the cyanopropyl and aminopropyl functionalities are popular for this use. These bonded phases are less subject to changing retention times of compounds because water is adsorbed from the mobile phase onto the stationary phase, a frequent concern when using bare silica packings for normal-phase separations. 

If two paths are possible, knowledge about the sample can guide the movement on the decision tree in Figure 4-1. Consider whether the differences between sample components are primarily steric or if the components differ in polarity or solubility. If steric, an adsorption packing is probably the best starting point otherwise a bonded-phase packing may be a better first choice. Your choice should be made based on convenience, personal prefer-... 

Once a mode of LC is chosen (Step 2), another uncertainty arises when deciding which specific packing should be used (Step 3). It is helpful to consider the available unbonded-phase (adsorption) and bonded-phase pack-... 

When used in the normal-phase mode, bonded-phase packings are stable to hydrolysis, have a shorter equilibration time to mobile phase changes than unbonded silica, and do not require water deactivation or humidified mobile... 

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