Bonded phase mobility

Euerby et al. [9] have systematically investigated the effects of the bonded phase, mobile phase, buffer type, field strength, pH, and temperature on the resolution of specific substituted barbiturates. Critical parameters for the optimization of efficiency of basic drugs by CEC (as for HPLC) include the nature of the sample solvent, pH, and concentration of ion-pair reagents, for example. 

Fig. 1 Separation of reversed-phase test mixture on a C22 bonded phase. Mobile phase 50 50 acetonitrile-water. Solutes 1 = benzene 2 = toluene 3 = ethylbenzene 4 = isopropylbenzene 5 = r-butylbenzene 6 = anthracene.

Fig. 2 Gradient separation of peptide mixture on a C30 bonded phase. Mobile phase, linear gradient from 25% to 45% A in 15 min. A = 0.1% trifluoroacetic acid (TFA) in 75 25 acetonitrile-water and B = 0.1% TFA in water. Solutes 1 = brady-kinin 2 = angiotensin III 3 = angiotensin I.

Column 250 X 4.6 Chromatography Sciences Co. octadecylsilane bonded phase Mobile phase MeCN THF buffer 300 75 625 (Buffer was 50 mM (NH4)H2P04 adjusted to pH 5.0 with 50 mM ammonium hydroxide.)... 

Support particle Nonpolar Interstitial area bonded phase (mobile phase)... 

Silica gel, per se, is not so frequently used in LC as the reversed phases or the bonded phases, because silica separates substances largely by polar interactions with the silanol groups on the silica surface. In contrast, the reversed and bonded phases separate material largely by interactions with the dispersive components of the solute. As the dispersive character of substances, in general, vary more subtly than does their polar character, the reversed and bonded phases are usually preferred. In addition, silica has a significant solubility in many solvents, particularly aqueous solvents and, thus, silica columns can be less stable than those packed with bonded phases. The analytical procedure can be a little more complex and costly with silica gel columns as, in general, a wider variety of more expensive solvents are required. Reversed and bonded phases utilize blended solvents such as hexane/ethanol, methanol/water or acetonitrile/water mixtures as the mobile phase and, consequently, are considerably more economical. Nevertheless, silica gel has certain areas of application for which it is particularly useful and is very effective for separating polarizable substances such as the polynuclear aromatic hydrocarbons and substances... 

Even with mobile-phase modifiers, however, certain polymer types cannot be run due to their lack of solubility in organic solvents. In order to run aqueous or mixed aqueous/organic mobile phases, Jordi Associates has developed several polar-bonded phase versions of the PDVB gels as discussed earlier. Figures 13.60 thru 13.99 detail examples of some polar and ionic polymers that we have been able to run SEC analysis of using the newer bonded PDVB resins. 

Scientific (Northbrook, IL) contain a silica support with a -y-glycidoxypropylsi-lane-bonded phase to minimize interaction with anionic and neutral polymers. The columns come in five different pore sizes ranging from 100 to 4000 A. The packing material has a diameter from 5 to 10 /cm and yields in excess of 10,000 plate counts. With a rigid silica packing material, the columns can withstand high pressure (maximum of 3000 psi) and can be used under a variety of salt and/or buffered conditions. A mobile phase above pH 8, however, will dissolve the silica support of the column (21). A summary of the experimental conditions used for Synchropak columns is described in Table 20.8. 

A computer program was compiled to work out the ray-tracing of UV detector of high performance capillary electrophoresis at the investigation of 5 and 6 (98MI59). The capacity factor of 5 at different temperature and at different mobile phase compositions was experimentally determined in bonded-phase chromatography with ion suppression (98MI15). 

To retain solutes selectively by dispersive interactions, the stationary phase must contain no polar or ionic substances, but only hydrocarbon-type materials such as the reverse-bonded phases, now so popular in LC. Reiterating the previous argument, to ensure that dispersive selectivity dominates in the stationary phase, and dispersive interactions in the mobile phase are minimized, the mobile phase must now be strongly polar. Hence the use of methanol-water and acetonitrile-water mixtures as mobile phases in reverse-phase chromatography systems. An example of the separation of some antimicrobial agents on Partisil ODS 3, particle diameter 5p is shown in figure 5. 

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. 

Bonded phases are the most useful types of stationary phase in LC and have a very broad range of application. Of the bonded phases, the reverse phase is by far the most widely used and has been applied successfully to an extensive range of solute types. The reverse phases are commonly used with mobile phases consisting of acetonitrile and water, methanol and water, mixtures of both acetonitrile and methanol and water, and finally under very special circumstances tetrahydrofuran may also be added. Nevertheless, the majority of separations can be accomplished using simple binary mixtures. 

It is seen that, providing the isomers are eluted at a (kf) value greater than about 2.0, the column will separate those solute pairs having separation ratios as low as about 1.03. This, however, assumes that the column is very well packed and is operated at about the optimum mobile phase velocity. In practice, a more realistic minimum separation ratio would be between 1.035 and 1.04. However, as it will be seen, the cyclodextrin bonded phases can easily provide separation ratios significantly greater than these values. This is achieved by... 

Other modes of LC operation include liquid-liquid partition chromatography (LLC) and bonded phase chromatography. In the former, a stationary liquid phase which is immiscible with the mobile phase is coated on a porous support, with separation based on partition equilibrium differences of components between the two liquid phases. This mode offers an alternative to ion exchange in the fractionation of polar, water soluble substances. While quite useful, the danger exists in LLC that the stationary phase can be stripped from the column, if proper precautions are not taken. Hence, it is typical to pre-equil-ibrate carefully the mobile and stationary phases and to use a forecolimn, heavily loaded with stationary phase 9). 

The precision in retention from injection to injection will often be better than 1%. Over longer periods of time such precision requires the following (a) good flow control from the pump (b) constant mobile and stationary phases and (c) temperature control of the column. The critical question of reproducibility from column to column is still a matter of concern, especially when dealing with the more sophisticated packing materials, e.g. small porous particles, bonded phases While frequently this reproducibility is quite good, workers should recognize that care must be exercised to achieve and/or maintain reproducible columns. Undoubtedly, with experience, this need not be a severe problem. 

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