Liquid Chromatography/Mass Spectrometry
Coupling HPLC with mass spectrometry allows analysis of mixture samples. It can provide information on individual compounds even when present in complex mixtures, including structural characterization through fragmentation and quantitation.
In LC/MS analysis, a sample is injected onto an LC column and separated into its various components. The components are eluted from the column and then pass into the MS through an electrospray interface. The data from the MS are then stored and processed.
The standard column in the lab is a 50×2.1 mm ID reverse phase C-18 column. The standard mobile phases are (A) 5% acetonitrile/95% water/0.1% formic acid and (B) 5% water/95% acetonitrile/0.1% formic acid. Ideally your chromatographic method would use this column and mobile phases. If you want to use a different column, it must be purchased and provided to the facility or the lab can purchase and bill you for the column. You may submit a method from the literature for a mixture similar to your own, or we can use a standard 30 minute or 1 hour linear gradient. If the standard column and mobile phases are not suitable, we can use 2.1-4.6 mm ID columns, columns with different packing materials (only reverse phase), or other mobile phases.
All chromatographic procedures developed should be free of non-volatile buffers, surfactants, salts, and non-volatile ion pair reagents. Mobile phases may have volatile buffers such as ammonium acetate in moderate concentration. Buffers such as phosphate, TRIS, and HEPES cannot be used. TFA, a common additive for reversed-phase separation, should be avoided or minimized since it will suppress electrospray ionization of the analytes.
For best results, you should provide optimized HPLC conditions, including the composition of the mobile phases, the gradient, the physical characteristics of the column, the flow rate, and the UV chromatogram.
