Bonnie A. Alden

Universal procedure for the assessment of the reproducibility and the classification of silica-based reversed-phase packings: II. Classification of reversed-phase packings

Abstract The tools developed for the testing of the reproducibility of several commercial packings have been used to study the differences and similarities of over 50 different commercial reversed-phase packings. The tests employed allow a characterization of the hydrophobicity of packings, the silanol activity at neutral and acidic pH, a differentiation between classical packings and packings with an embedded polar functional group, and a differentiation between C18 and C8 packings.

Universal procedure for the assessment of the reproducibility and the classification of silica-based reversed-phase packings : I. Assessment of the reproducibility of reversed-phase packings

A method is described that has been used since 1985 to assess the quality and reproducibility of several popular reversed-phase packings. Both the precision of the method and the reproducibility of the packing materials are described. The reproducibility of the newer packings surpasses that of the older packings. In addition, improved results are achieved today for the packings that existed in 1985.

Systematic Study of Chromatographic Behavior vs Alkyl Chain Length for HPLC Bonded Phases Containing an Embedded Carbamate Group

A series of HPLC bonded phases containing an internal carbamate group were studied by changing the terminal N-alkyl group from C(8)H(17) to C(18)H(37) in increments of two methylene units, i.e., C(8), C(10), C(12), C(14), C(16), and C(18). Each material was prepared via bonding of silica with the respective 3-(chlorodimethylsilyl)propyl N-alkylcarbamate silane. The materials were compared under reversed-phase conditions using a test mixture of nonpolar, polar, and basic compounds in a 65:35 (v/v) methanol/20 mM KH(2)PO(4)/K(2)HPO(4), pH 7, mobile phase. Retention factors were found to generally increase from the C(8) length to the C(12)-C(16) lengths but decreased for the C(18) length. Retention factors were also measured as a function of three ligand surface concentrations for the C(12), C(14), and C(18) materials. In this study, retention generally decreased with increasing surface concentration, especially for the C(18) chain length. Changes in particle surface area and porosity caused by bonding did not fully account for the observed changes in retention factors. Peak shapes for the basic analytes propranolol and amitriptyline were also studied as a function of N-alkylcarbamate chain length and surface concentration. Tailing factors were unaffected by chain length and only weakly dependent on surface concentration. By comparison, tailing factors decreased significantly as surface concentration increased for a set of conventional C(18) alkyl packings.

Dependence of cyano bonded phase hydrolytic stability on ligand structure and solution pH.

As part of our program to develop more stable cyano (CN) high-performance liquid chromatography (HPLC) column packings, we have evaluated hydrolytic stability as a function of ligand connectivity, chain length, and side group steric protection and the pH of the mobile phase. Three accelerated tests were used to evaluate stability: (1) A non-HPLC screening test measuring carbon loss in refluxing MeOH-100 mM KH2PO4 pH 4.5 (1:1, v/v) solution; (2) a continuous flow HPLC test measuring capacity factor maintenance in 1% trifluoroacetic acid in water (pH 1.02) at 80 degrees C; and (3) a continuous flow HPLC test measuring column efficiency maintenance in 50 mM triethylamine in water (pH 10.00) at 50 degrees C. The stability of the CN phases was found to be dependent on both ligand chemical structure and the pH of the test conditions. The starting screen test of intermediate pH was least able to differentiate the CN phases based on structure, because two different degradation mechanisms appear to offset each other (acid induced siloxane bond cleavage vs. base induced silica dissolution). A trifunctional and a sterically protected CN phase were notably stable under the acidic test conditions, but had poor stability under basic conditions. Conversely, chain extension afforded poor stability under acidic conditions, but did afford improved stability at higher pH. In total, the data indicate that good CN column stability can be achieved by using a trifunctional or a sterically protected phase in acidic mobile phases. However, as mobile phases of intermediate or higher pH are employed, shorter column lifetimes can be expected due to an accelerated dissolution of the underlying silica substrate. Materials were also compared chromatographically using a mixture of non-polar, polar, and basic analytes under reversed-phase conditions.

High-resolution peptide mapping separations with MS-friendly mobile phases and charge-surface-modified C18.

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.

3 HPLC columns and packings

Abstract In this chapter on HPLC columns, we are discussing both the surface chemistry of a packing as well as column design and performance. In the section that covers column chemistry, we cover modern options of base materials as well as the commonly used approaches towards the surface chemistry of a packing. Specific subsections are dedicated to the selectivity of reversed-phase packings, HILIC, monolithic structures, and the reproducibility of modern packings. In the section on speed and resolution, we familiarize the reader with the principles of how to choose a column. In the section on specialty columns, we cover briefly preparative chromatography and columns with a very small diameter.

4 – HPLC Columns for Pharmaceutical Analysis

This chapter deals with the properties of high-pressure liquid chromatography columns. It is divided into two sections: column physics and column chemistry. In the section on column physics, we discuss the properties that influence column performance, such as particle size, column length and column diameter, together with the effect of instrumentation on the quality of a separation. In the section on column chemistry, we examine in depth the surfaces of modern packings, as well as the newer developments such as zirconia-based packings, hybrid packings or monoliths. We have also included a short section on hydrophilic interaction chromatography, a technique for the analysis of polar compounds that is drawing interest again in the pharmaceutical industry. Finally, we review what is currently understood about the selectivity of reversed-phase columns.