A typical LC/MS analysis begins with the liquid chromatograph (LC) separating a mixture into its chemical components. The LC pump produces a solvent stream (the mobile phase) that passes through an HPLC column (containing the stationary phase) under high pressure. An autosampler introduces an aliquot of sample into this solvent stream. As the solvent stream passes through the LC column, the sample separates into its chemical components. The rates at which the components of the sample elute from the column depend on their relative affinities to the mobile phase and the stationary phase.
As the separated chemical components exit the LC column, they pass through a sample transfer line and enter the mass spectrometer for desolvation, ionization, and analysis. As the MS analyzes the ionized components and determines the mass-to-charge ratio (m/z) and relative intensity of each ion, it sends a data stream to the data system computer. In addition to supplying information about the m/z values of ionized compounds, the MS can also supply structural and quantitative information by performing MSn experiments.
With a syringe pump and an optional divert/inject valve, you can introduce samples into the MS using the methods as described in the following table.
Methods of sample introduction into the MS
Method | Description |
|---|---|
Direct infusion | A syringe pump is connected directly to the atmospheric pressure ionization (API) source of the MS. |
NOTE This method provides no chromatographic separation of components in the sample before they pass into the MS. | |
High-flow infusion | Uses a union Tee to combine the flow from the syringe pump with the flow from an LC pump. See Set up the Plumbing for High-flow Infusion (without an autosampler). |
NOTE This method provides no chromatographic separation of components in the sample before they pass into the MS. | |
Manual loop injection | Connects a sample loop, a needle port fitting, and an LC pump to the divert/inject valve. After you fill the loop with sample, switch the position of the divert/inject valve to place the contents of the sample loop in the path of the solvent flow produced by the LC pump. See Plumbing Setups for Manual Loop Injections (flow-injection analysis). |
TIP
For information about setting up the various sample introduction methods, see To Connect the Inlet Plumbing.
The MS consists of an API source, ion optics, two mass analyzers, and an ion detection system. All are enclosed in a vacuum manifold, except for part of the API source.
Ionization of the sample takes place in the API source. The specific method that you use to ionize the sample is referred to as the ionization technique. The ion optics transmit the ions produced in the API source into the mass analyzer to determine their m/z values. The polarity of the electric potentials applied to the API source and ion optics determines whether positively charged ions or negatively charged ions are transmitted to the mass analyzer. Certain lenses in the API source and ion optics act as gates to start and stop the transmission of ions from the API source to a mass analyzer. An Automatic Gain Control™ (AGC) process controls the function of these lenses and sets them to transmit the optimum number of ions to the mass analyzer.
Each sequence of loading a mass analyzer with ions followed by mass analysis of the ions is called a scan. The ability to vary not only the ionization and ion polarity modes, but the scan mode and scan type, provides greater flexibility in the instrumentation for solving complex analytical problems. You can set up data acquisition methods for the MS to analyze positively or negatively charged ions or to switch between these polarity modes during a single run.
The data system serves as the user interface to the MS, autosampler, LC pump, and syringe pump.