Bonded-phase packings. HPLC

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... 

Kirkland, J. J., Glajch, J. L. and Farlee, R. D., Synthesis and Characterization of Highly Stable Bonded Phases for HPLC Colirmn Packings, Anal. Chem., 61 2—11, 1989. 

Bonded-phase packings In HPLC, a support medium to which a liquid stationary phase is chemically bonded. 

Manuhicturers of silica-based HPLC packings have spent much effort learning to understand and mitigate the influence of silanols, and much improvement has been made with newer bonded-phase packings. For a more detailed discussion, see Chapter 10. 

Micropacked columns are available with most of the bonded-phase packings used in HPLC. Porous and non-porous silica particles are optionally functionalized with covalently ound silanes or other strongly adsorbed materials. ALkyl-bonded silicas produce separations, generally based on solute volatility, but with the potential for selectivity differences based on interaction with silanol groups. Underivatized silica is popular for petroleum separations of aliphatic and aromatic hydrocarbons. Silver-ion-containing silica columns are selective for olefin separations. Huoroalkyl-bonded... 

More recently, several reports have appeared which describe the preparation of HPLC columns which contain CD chemically bonded to silica gel [15-18]. Of these, there are presently two types. The first consists of CD bonded to the silica via amide or amine bonds [15,16] while the second contains no nitrogen linkages [17,18]. This review article summarizes our chromatographic work to date with the latter type of CD bonded phases. In particular, we demonstrate the successful HPLC separation of enantiomers, epimers, cis-trans and other structural isomers as well as important classes of routine compounds by use of a P- or y-CD bonded phase. The obtained chromatographic separations are compared to those obtained on the more conventional normal or reversed phase packings. Additionally, the effect of changes of the pertinent chromatographic variables (such as flow rate, temperature, and solvent composition of the mobile phase) upon the separations are described. Lastly, a brief prospectus on the future of CD bonded phases in HPLC is presented. 

Bonded-phase packings arc classified as reversed phase when the bonded coating is nonpolar in character and as normal phase when the coating contains polar functional groups. It has been estimated that more than three quarters of all HPLC separations are currently performed in columns with reversed-phase packings. The major advantage of reversed-phase separations is that water can be used as the mobile phase. 

In NPLC, which refers to the use of adsorption, i.e. liquid-solid chromatography (LSC), the surface of microparticulate silica (or other adsorbent) constitutes the most commonly used polar stationary phase normal bonded-phase chromatography (N-BPC) is typified by nitrile- or amino-bonded stationary phases. Silica columns with a broad range of properties are commercially available (with standard particle sizes of 3, 5 and 10 im, and pore sizes of about 6-15nm). A typical HPLC column is packed with a stationary phase of a pore size of 10 nm and contains a surface area of between 100 and 150m2 mL-1 of mobile phase volume. 

Since selectivity in HPLC involves both the stationary and mobile phases [5-9,58-60], it is important to note that the solvent strength of the mobile phase, as compared to the stationary phase, (composed of mobile-phase components reversibly retained by the bonded phase and silica support) determines the elution order or k of the retained components. Unfortunately, the columns with the same stationary phase can exhibit significant variabilities from one manufacturer to another and even from the same manufacturer [5-8]. Based on discussions heard at various scientific meetings, this situation has not changed much. Variabilities can occur in the packing process even where all other conditions are supposedly constant. These factors have to be considered prior to developing an understanding as to how separations occur in HPLC. 

When such microparticulate-bonded-phases are packed compactly into a column by means of a suitable device, the small size of these particles offers a significant resistance to solvent flow therefore, the mobile phase has to be pumped through the column under a high positive pressure. For an analytical HPLC, the mobile-phase is pumped through the column at a flow rate of 1-5 cm3, min-1. 

Reverse-phase HPLC can be used for the separation of peptides and proteins. Smaller peptides (less than 50 amino acid residues) may be satisfactorily separated on octadecylsilane (C-18) bonded phases whereas for adequate recovery of larger molecules, tetrylsilane (C-4) or octylsilane (C-8) is recommended. Porous column packing with gel permeation and reverse phase properties is usually required for proteins with relative molecular masses greater than 50 000. 

Column—a tube that contains packed sorbents. Typical HPLC columns are stainless-steel tubes packed with silica-based bonded phases. 

Depending on the particular method of polymer HPLC, is defined in different ways. It is the total volume of pores, Vp, in a porous packing but it can be also related to the total surface of packing (mostly to the surface situated within the pores) or to the effective volume of bonded phase. The volume of pores is relatively well defined in the case of many packings applied in polymer HPLC and plays an especially important role in the exclusion-based separations (Sections 16.3.3, 16.3.4, and 16.4.1). The exclusion processes, however, play an important role in the coupled techniques of polymer HPLC (Section 16.5). In the latter cases, the surface of packings and the effective volume of bonded phase are to be taken into account. In some theoretical approaches also, surface exclusion is considered. 

Similar to other coupled methods of polymer HPLC, for example, LC CC (Section 16.5.2), the choice of the column packing and the mobile phase components for EG-LC depends on the retention mechanism to be used. Adsorption is preferred for polar polymers applying polar column packings, usually bare silica or silica bonded with the polar groups. The eluent strength controls polymer retention (Sections 16.3.2 and 16.3.5). The enthalpic partition is the retention mechanism of choice for the non polar polymers or polymers of low polarity. In this case, similar to the phase separation mechanism, mainly the solvent quality governs the extent of retention (Sections 16.2.2, 16.3.3, and 16.3.7). It is to be reminded that even the nonpolar polymers such as poly(butadiene) may adsorb on the surface of bare silica gel from the very weak mobile phases and vice versa, the polymers of medium polarity such as poly(methyl methacrylate) can be retained from their poor solvents (eluents) due to enthalpic partition within the nonpolar alkyl-bonded phases. 

Alumina and silica columns, formerly used for PAH separation, were later replaced with chemically bonded phases [685]. RP-HPLC based on the use of C18 columns is nowadays the most popular mode for PAH separation, and specially designed colnmns from different vendors are commercially available. Colnmns of 100-250cm length (3.0-4.6mm i.d.), packed with 3-5pm particles. 

Improved separation of natural oil TGs using short columns packed with 3-//m alkyl bonded-phase particles was reported by Dong and DiCesare (88). The HPLC columns used were HS-3 high-speed columns packed with 3-/um C18 bonded-phase particle (100 X 4.6-mm ID) with a column void volume of ca. 0.8 ml and efficiencies in the range of 13,000-15,000 theoretical plates (measured under optimized conditions) and HS5 C,8 columns (125 X 4.6-mm ID packed with 5-yttm particles). Two detectors were used a modified refractive index detector having an 8-/rl flow cell and 0.007-in. ID inlet tubing and a variable-wavelength UV/visible detector. 

Imazalil, OPP, and TBZ residues were determined using an HPLC system, consisting of a ternary HPLC pump attached to 250 X 4.6-mm-ID Hichrom RPB 5-/tm column with a 10 X 3.2-mm-ID guard cartridge column. The packing material of both columns was base-deactivated silica, fully endcapped with a bonded phase of Cg/C]8. The mobile phase was methanol/water at a flow rate of 1 ml/min at ambient temperature. Imazalil in the eluate was detected with UV detector at 204 nm a fluorescence detector was used to monitor OPP (excitation 285 nm, emission 350 nm) and TBZ (excitation 296 nm, emission 350 nm) (47). 

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