Normal Phase HPLC

Chromatography is the separation of components in a mixture based on their chemical nature. Chromatography can be performed in either liquid (HPLC) or gas (GC) forms. Liquid chromatography, specifically High Pressure Liquid Chromatography (HPLC), involves a stationary phase (sorbent), a mobile phase (liquid) and the analyte or compounds of interest. Analytes or compounds of interest are injected onto an HPLC column (packed with sorbent that contains a variety of chemistries) in mobile phase. As the mobile phase, which is carrying the analyte or compounds of interest, travels across the stationary phase, the analyte or compounds of interest separate from each other as they interact with the stationary phase, each eluting from the column at various times. Gas chromatography (GC) is similar to HPLC in that is utilized with working analytes or compounds of interest that can be vaporized without decomposing. In GC, gas is used instead of a liquid (like in HPLC). As analytes or compounds of interest travel in the gas mobile phase and interact with the stationary phase, eluting at various times.

Luna NH2 (amino) columns are shipped in 99:1 hexane:acetonitrile. If the column is to be used in reversed phase or HILIC conditions, it is recommended to flush with IPA at low flow rate (10 column volumes) prior to introducing water:acetonitrile or water:methanol mixtures.

The cleaning procedure for Luna NH2 columns will depend on the separation mode it is being for. Before starting any kind of cleaning procedure, make sure your in-column solvent or mobile phase is miscible with the recommended cleaning solvents. Also, flow-rates should be 1/5-1/2 of the typical flow-rate. To estimate column volume, use the following equation: V=πr²L V= column volume in mL r= column radius in cm L= column length in cm See the cleaning protocols below. NH2 for Normal Phase: Rinse with 10 Column Volumes each of: Chloroform Isopropanol Methylene Chloride Mobile Phase NH2 for Reversed Phase: Rinse with 10 Column Volumes each of: 95 % Water/5% Acetonitrile (for buffer removal) THF 95% Acetonitrile/5% Water Mobile Phase NH2 for HILIC or Ion-Exchange*: Rinse with 10 Column Volumes each of: 50/50 Organic (e.g. Acetonitrile or Methanol)/20mM ammonium bicarbonate pH 10 (to clean ionically bound compounds) Water Mobile Phase *This column cleaning procedure should only be done infrequently, as repeated exposure to high pH solutions can cause silica dissolution, resulting in peak shape issues

The expected back pressures generated by a Kinetex core-shell column should be approximately what is observed with a column packed with fully porous particles of the same diameter. The observed backpresure for a given method will depend on column dimension, flow-rate and solvent composition. That said, backpressures are typically less than 250 bar. Kinetex 5 µm columns are rated to withstand pressures up to 600 bar.