The characterization and evaluation of three novel 5-microm HPLC column packings, prepared using ethyl-bridged hybrid organic/inorganic materials, is described. These highly spherical hybrid particles, which vary in specific surface area (140, 187, and 270 m(2)/g) and average pore diameter (185, 148, and 108 A), were characterized by elemental analysis, SEM, and nitrogen sorption analysis and were chemically modified in a two-step process using octadecyltrichlorosilane and trimethylchlorosilane. The resultant bonded materials had an octadecyl surface concentration of 3.17-3.35 micromol/m(2), which is comparable to the coverage obtained for an identically bonded silica particle (3.44 micromol/m(2)) that had a surface area of 344 m(2)/g. These hybrid materials were shown to have sufficient mechanical strength under conditions normally employed for traditional reversed-phase HPLC applications, using a high-pressure column flow test. The chromatographic properties of the C(18) bonded hybrid phases were compared to a C(18) bonded silica using a variety of neutral and basic analytes under the same mobile-phase conditions. The hybrid phases exhibited similar selectivity to the silica-based column, yet had improved peak tailing factors for the basic analytes. Column retentivity increased with increasing particle surface area. Elevated pH aging studies of these hybrid materials showed dramatic improvement in chemical stability for both bonded and unbonded hybrid materials compared to the C(18) bonded silica phase, as determined by monitoring the loss in column efficiency through 140-h exposure to a pH 10 triethylamine mobile phase at 50 degrees C.
Several chromatographic test procedures are in use for the characterization of commercially available packings. The results of the test procedure used in our laboratory are updated and further refined. Two well-defined physico-chemical properties of packings can be derived from this test procedure, the hydrophobicity of a packing and the silanophilic activity at pH 7. In addition, our method is unique in its ability to differentiate between classical packings and packings with incorporated polar groups. We can also distinguish CN packings or fluorinated packings. Our database now includes over 100 commercially available packings. The ability of the test to cleanly discriminate between different classes of packings has been strengthened. Limited comparisons with other published tests are now possible as well, and the results of these comparisons are discussed.
Ionic analytes, such as peptides, can be challenging to separate by reverse-phase chromatography with optimal efficiency. They tend, for instance, to exhibit poor peak shapes, particularly when eluted with mobile phases preferred for electrospray ionization mass spectrometry. We demonstrate that a novel charged-surface C18 stationary phase alleviates some of the challenges associated with reverse-phase peptide separations. This column chemistry, known as CSH (charged-surface hybrid) C18, improves upon an already robust organosilica hybrid stationary phase, BEH (ethylene-bridged hybrid) C18. Based on separations of a nine-peptide standard, CSH C18 was found to exhibit improved loadability, greater peak capacities, and unique selectivity compared to BEH C18. Its performance was also seen to be significantly less dependent on TFA-ion pairing, making it ideal for MS applications where high sensitivity is desired. These performance advantages were evaluated through application to peptide mapping, wherein CSH C18 was found to aid the development of a high-resolution, high-sensitivity LC-UV-MS peptide mapping method for the therapeutic antibody, trastuzumab. From these results, the use of a C18 stationary phase with a charged surface, such as CSH C18, holds significant promise for facilitating challenging peptide analyses.
Reversed-phase packings with an incorporated polar group, specifically a carbamate group, are compared with classical packings and the results are discussed. In this study, the underlying silica and the surface coverage are identical, allowing for a simple and unequivocal comparison of the sorbent properties that originate solely in the nature of the ligancl. The polar group contributes to improved peak shapes for acidic, basic and zwitterionic analytes and changes the selectivity of a separation compared to classical bonded phases. A chromatographic technique that allows a discrimination bef,,veen classical bonded phases and those with an incorporated polar group is described. In addition, phases with an embedded polar group can be used without difficulty in mobile phases with a very high water content, which makes them suitable for the separation of very polar analytes. ExperimentalColumns and instruments used in the studies shown here were from Waters Corporation. Packing materials used were IxBondapak T M C1s, Symmetry | Cs and C1s, SymmetryShield | RPs and RP1s, and XTerra | RPs and MS Cs packings, ixBondapak T M C1s, Symmetry | Cs and C1s, and XTerra | MS Cs are packings with a clas-Original
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