The Application Notebook
One problem frequently encountered in LC–MS is the appearance of mass peaks, which appear totally unrelated to the samples run - "ghost" mass peaks. It is impossible to differentiate whether these signals come from an unknown component in the sample co-eluting with a known peak, or from an impurity in the mobile phase or from some residual contamination "bleeding" from the column.
One problem frequently encountered in LC–MS is the appearance of mass peaks, which appear totally unrelated to the samples run — "ghost" mass peaks. It is impossible to differentiate whether these signals come from an unknown component in the sample co-eluting with a known peak, or from an impurity in the mobile phase or from some residual contamination "bleeding" from the column.
To solve this problem, a three-step procedure is proposed for washing columns and choosing solvents for use in LC–MS measurements. This procedure also increases ionization efficiency and thereby increases both sensitivity and reproducibility of LC–MS measurements.
Step 1: Procedure for "Washing" the HPLC Column
A clean HPLC column is an essential pre-requisite for good LC–MS results. However, trace residues of organic compounds are nearly always present on reversed-phase HPLC columns. Residues may come from previously injected samples, from solvent impurities or from the silanes bonded to the silica surface. These residues are typically eluted very slowly from the column by the mobile phase. This phenomenon is called "column bleeding" and can give rise to unwanted background detector signals and can be very disturbing in LC–MS.
To minimize or even eliminate this "column bleeding", a simple procedure is to pump iso-propanol with 0.1% formic acid at room temperature for one hour through the column. For a column with 3 mm i.d., 0.5 mL/min flow-rate is appropriate. It is important that the column is not connected to the mass spectrometer to avoid contaminating the ion source.
Step 2: The Choice of Solvent Grade for the Mobile Phase
HPLC gradient or isocratic grade solvents are generally specified for use with UV detectors, sometimes with fluorescence detectors, but are not specified for use in LC–MS. Trace organic impurities may not absorb in UV at the wavelength used, but may be ionized in the mass spectrometer, thereby giving rise to background signals which appear in the mass spectra, making the spectra complex and difficult to interpret. Such impurities "compete" with the sample components in the ionization process and this competition causes "ionization suppression". The ionization efficiency is reduced and sensitivity is lost. Figure 2 shows a typical mass spectrum with low signal-to-noise ratio using standard reagent grade acetonitrile.
Figure 3 shows the same spectrum measured using LC–MS grade acetonitrile (Merck LiChrosolv hypergrade 1.00029). The ionization efficiency is increased more than 10-fold. This is a critical performance improvement. In addition, the intensity of background signals is negligible and all mass signals* can be directly correlated to the target compound, ethyl paraben.
*(mass 167.1 = M+H, mass 189.0 = M+Na, mass 139.1 = fragment protonated p-hydroxybenzoic acid, mass 121.2 = protonated fragment)
Step 3: Equilibrating the HPLC Column
Finally the column must be re-equilibrated with the mobile phase. Best results are obtained when two blind gradients (without sample injection) are run prior to analysing samples.
By application of a simple column washing procedure and use of LC–MS specified solvent, LC–MS results can be improved in three ways:
The method has been verified with Chromolith RP-18e (monolithic silica) and Purospher STAR RP-18e 3 μm and 5 μm HPLC columns.
1. Solvents available from Merck in LC–MS specified quality: Acetonitrile — LiChrosolv hypergrade 1.00029 Methanol — LiChrosolv hypergrade 1.06035
2. Chromolith RP-18e columns are made from high-purity metal-free monolithic silica and used for fast LC–MS Purospher STAR RP-18e columns are made from high-purity metal-free 3 μm and 5 μm silica particles
Alexander Kraus, Rod McIlwrick, Gisela Jung and Ines Hädrich, Merck KGaA, Darmstadt, Germany.
Frankfurter Strasse 250, D-64293 Darmstadt, Germany
Fax: +49 6151 72 6080
Websites: www.merck.de, www.chromatography.merck.de.