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 rate at which the components of the sample elute from the column depends 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 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.
When the system setup includes a syringe pump and divert/inject valve, there are three additional ways to introduce samples into the MS, as described in the following table.
Method |
|
|---|---|
Direct infusion | Connect the syringe pump directly to the API source of the MS. |
High-flow infusion | Use a union Tee to combine the flow from the syringe pump with the flow from an LC pump. |
Manual loop injection | Connect a sample loop, a needle port fitting, and an LC pump to the divert/inject valve. After you fill the sample loop with sample, switch the position of the divert/inject valve, which places the contents of the sample loop in the path of the solvent flow produced by the LC pump. |
NOTE
Analysis by direct infusion or flow injection provides no chromatographic separation of components in the sample before they pass into the MS.
TIP
For information about setting up the various sample introduction methods, see Sample introduction techniques.
The MS consists of an API source, the ion optics, a triple-stage mass analyzer, 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. 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. Each sequence of loading a mass analyzer with ions followed by mass analysis of the ions is called a scan.
The data system serves as the user interface to the MS, autosampler, LC pump, and syringe pump. Refer to the data system Help for information about its instrument control and data-processing features.
NOTE
For a list of ion sources that are compatible with your TSQ Series II mass spectrometer, see Compatible ion sources.
For information about the heated-electrospray (H-ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) techniques, refer to the user guide for your high-flow ion source.
For information about the nanoelectrospray ionization (nanoESI or NSI) technique, refer to the user guide for your nanoflow ion source.
The mass spectrometer’s triple-stage mass analyzer performs either one stage or two stages of mass analysis:
- The TSQ Series II mass spectrometer operates as a conventional MS system with one stage of mass analysis. The ion source ionizes the sample, and mass filtering of the ions occurs in the first rod assembly. The second and third rod assemblies transmit the resulting mass-selected ions to the ion detection system.
- Note: You can also use the instrument as a single-stage MS by transmitting the ions through the first and second rod assemblies followed by mass analysis in the third rod assembly.
–or–
- The TSQ Series II mass spectrometer operates as a tandem MS system with two stages of mass analysis. The ion source ionizes the sample, and mass filtering of the ions occurs in the first rod assembly (Q1). In this case, however, mass-selected ions exiting the first rod assembly collide with an inert gas in the second rod assembly (Q2) and fragment to produce a set of ions known as product ions. (A chamber called the collision cell surrounds the second rod assembly. The MS pressurizes the collision cell with an inert gas.) The third rod assembly (Q3) performs mass filtering on the product ions to detect selected ions. Two stages of mass analysis yield far greater chemical specificity than a single analysis stage because of the system’s ability to select and determine two discrete but directly related sets of masses.
During a scan, the first stage of mass analysis elucidates the elemental compositions of pure organic compounds and the components within mixtures. Furthermore, in a scan sequence that involves a second stage of mass analysis, the MS can fragment and separate each ionic fragment of a molecule formed in the ion source to elucidate the structure of the molecule.
The two stages of mass analysis are ideal for very selective and sensitive analysis by reducing chemical noise in the final mass spectrum.
The MS uses several different scan modes and scan types to filter, fragment, or transmit ions in the mass analyzer, including the ionization and ion polarity modes. This ability to vary the scan mode and scan type provides greater flexibility in the instrumentation for solving complex analytical problems.