Ascentis^® Express 120 Å C18 pH+ (2.7 µm) HPLC Column

Ascentis^® Express 120Å C18 pH+ is a high-performance liquid chromatography column based on the Fused-Core^® particle design, enabling high-throughput applications. The Fused-Core^® particle provides a thin porous shell of high-purity silica surrounding a solid silica core. This particle design exhibits very high column efficiency due to the shallow diffusion paths in the 0.5 µm deep porous shell and the small overall particle size of 2.7 µm. Ascentis^® Express 120Å C18 pH+ has a surface modification that leads to alkaline resistance, which provides a stable, reversed-phase packing that can be used for separations run under high pH mobile phase conditions. It is particularly useful for improving peak shape and load tolerance of basic compounds at elevated pH, without detrimental effects to acids or neutrals.

The Ascentis^® Express 120Å C18 pH+ bonded phase is a dimethyloctadecylsilane that has been specifically designed for applications requiring mobile phases that are mixtures of methanol and water or acetonitrile and water with pH ≥ 8 mobile phase buffers or additives. Using elevated temperatures (e.g., 40 – 60 ºC) will reduce the viscosity of the mobile phase and allow the use of faster flow rates and lower column pressure for high sample throughput. Gradient elution may start at 5% organic and then increase to 100% organic component or lower, given the hydrophobicity of the analytes being separated. Ascentis^® Express 120Å C18 pH+ columns are highly suited for the reversed-phase separation of basic, acidic, and neutral compounds. Additional information on solvent selection and separation techniques can be found in Chapters Six, Seven, and Eight, Practical HPLC Method Development, Second Edition, L.R. Snyder, J.L. Glajch, and J.J. Kirkland, (John Wiley & Sons, 1997). Volatile buffers such as 10-20 mM ammonium acetate and ammonium hydroxide are recommended for use with MS detection. Ammonium bicarbonate and organic buffers such as TRIS are recommended for UV detection. Phosphate and borate may be used for UV detection, but may be harsher on LC instrumentation. For more information on the impact of buffer on column lifetime, see Journal of Chromatography A, 728 (1996) 259-270.

To maximize column life, ensure that samples and mobile phases are particle-free. The use of guard columns or an in-line filter with 0.5 µm porosity between the sample injector and the column is highly recommended. Should the operating pressure of the column suddenly increase beyond normal levels, reversing the flow direction of the column may be attempted to remove debris on the inlet frit. To remove strongly retained materials from the column, flush the column in the reverse direction with very strong solvents such as 100% of the organic component of the mobile phase in use. A mixture (95/5 v/v) of dichloromethane and methanol is often effective at this task. Extreme cases may require the use of very strong solvents such as dimethylformamide (DMF) or dimethylsulfoxide (DMSO). Column Storage: Long-term storage of silica-based, reversed-phase columns is best in 100% acetonitrile. Columns may be safely stored for short periods (up to 3 or 4 days) in most common mobile phases. However, when using buffers, itis best to protect both the column and the HPLC equipment and remove the saltsby flushing the column with the same mobile phase without the buffer (e.g., when using 60/40 MeCN/buffer, flush the column with 60/40 MeCN/H2O)to eliminate any danger from corrosion from the salts while providing rapidre-equilibration of the column with the original mobile phase. Before storing the column, the end-fittings should be tightly sealed with the end-plugs that came with the column to prevent the packing from drying.

Ascentis is a registered trademark of Merck KGaA, Darmstadt, Germany

Fused-Core is a registered trademark of Advanced Materials Technology, Inc.