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 identification by molecular weight, structure characterization through fragmentation, and quantitation. Obviously, it provides more specific information about a compound than optical detectors.
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. We can perform ESI-LC/MS of tryptic digests and protein mixtures or any compounds which produce ions by ESI.
The standard column in the lab is a 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 and be developed in your lab prior to submitting your sample. If you are unable to develop your own method, 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 4.6 mm ID columns, columns with different packing materials, or other mobile phases. We can also use your own column and 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.
LC/MS Instrument: Q-Tof. Sample size: 100-500 nmole of a tryptic digest