Coupling Methods in Mass Spectrometry

Part 1: GC-MS, HPLC-MS, CE-MS and TLC-MS

  • Fig. 1: Schematic representation of the ionization techniques A) electron impact ionization (EI), B) field desorption (FD/FI/LIFDI), C) electrospray ionization (ESI) and D) matrix-assisted laser desorption ionization (MALDI). A description of the processes can be found in the text.
  • Fig. 2: Presentation of the fields of application of the most common ionization techniques regarding polarities and molecular weights of the analytes.

Until the 1990s, the coupling of “mass-selective detectors” with separation techniques was limited to gas chromatographs. Since then, the development of new ionization techniques has greatly extended the coupling possibilities to all other common separation techniques. Coupling methods in mass spectrometry have become indispensable in chemical, environmental, food, pharmaceutical and bioanalytical applications.

 

Why Coupling Separation Techniques and Mass Spectrometry?

Chromatography and electrophoresis are ideally suited for the separation of complex sample mixtures. Especially with very complex samples or even unknown compounds, the information content of e.g. UV-Vis in liquid- or flame ionization detectors in gas-chromatography is often not sufficient to identify compounds reliably.
Mass spectrometers on the other hand provide information on the molecular composition and ideally also on the structure of chemical compounds. Powerful mass spectrometers are able to determine masses very accurately and with high resolution, so that at least for small molecules, sum formulas can be obtained directly. Fragment ion spectra can also be used to obtain structural information. With complex mixtures, however, ion suppression effects and possibly also disturbing signal superpositions can occur. Especially with electron impact ionization (EI), a reliable assignment of the fragmentation signals caused by the ionization technique is usually not possible for mixtures of substances. In addition, with limited sample quantities and targeted fragmentation of individual components, the available measurement time is generally not sufficient to fragment all components and the isolation width can also be limiting in case of isobaric compounds.
It is therefore only logical and consistent to combine both methods and thus eliminate the respective limitations of the individual methods.
 
Coupling Gas Chromatography with Mass Spectrometry (GC-MS)
The gas chromatography-mass spectrometry coupling (GC-MS) is historically the oldest of the coupling techniques presented in this article, since the ionization techniques electron impact ionization (EI), chemical ionization (CI) and field ionization (FI) suitable for GC-MS coupling have been commercially available for many decades [1].

All three techniques mentioned, ionize the analyte molecules inside the instrument, i.e. in the high vacuum of the mass spectrometer, and assume - like gas chromatography itself - that the analytes can be vaporized undecomposed, so that the GC-MS coupling is more suitable for hydrophobic or derivatized hydrophobized small molecules [2]. The outlet of the separating capillary of the GC is led gas-tight into the ionization chamber of the mass spectrometer via appropriate seals. The carrier gas continuously emerging from the GC column into the ionization chamber must be continuously pumped out in order to maintain the vacuum in the mass spectrometer, so that the use of the capillary columns, which are nowadays mostly used and operated with low flow rates anyway, is appropriate.

 
The Ionization Techniques EI, CI and FI
During electron impact ionization (EI), a hot cathode emits electrons. In the ionization housing, an electron beam forms between the glow cathode and the capture anode. When analyte molecules pass the electron beam, an electron can be knocked out of the outer shell of an analyte molecule, resulting in radical cations. At a standardized electron energy of 70 eV, formed ions are unstable and decay very reproducibly into characteristic fragments which are used for the automated identification of analytes by means of spectrum libraries. GC-MS can be used to easily and reliably identify and quantify compounds present in the used database. The frequently used NIST spectra library currently contains more than 250,000 entries.
Chemical ionization (CI) works analogously to EI, with the exception that it is not the analyte molecules themselves that are primarily ionized, but reactant gas molecules. Methane, isobutane or ammonia can be used as reactant gas. Between ionized reactant gases and analyte molecules, charge transfer takes place, usually by protonation (positive ion mode) or deprotonation (negative ion mode). Especially the negative chemical ionization is extremely sensitive. In 1992, McLafferty and Michnowicz succeeded in a GC-MS experiment using negative chemical ionization to detect a quantity of octafluoronaphthalene corresponding to about 200,000 molecules [3]. In contrast to EI, CI produces significantly fewer fragment ions.
Virtually no fragmentation occurs during field ionization (FI), which is a variant of field desorption (FD). The source housing contains a carbon-activated metal thread to which a high voltage is applied. At the tips of the fine branches of the carbon dendrites (whiskers), very high field strengths are formed, which ultimately lead to the tunneling of individual electrons from analyte molecules in the immediate vicinity of the filament and thus to the gentle ionization of the analytes [4,5]. FI unfortunately has the major disadvantage that it is about three orders of magnitude less sensitive than EI and CI.
 
Ionization Techniques for the Coupling of HPLC, CZE, and TLC with Mass Spectrometry
Electrospray ionization (ESI) involves pressing the analytes in solution through a capillary. When high voltage is applied – depending on the flow rate approx. 1.5–5 kV – a spray of charged droplets is formed [7]. Solvents are removed from the droplets by continuous evaporation processes, so that the charge density increases. Since charges of the same name repel each other, the droplets are split into smaller droplets at a certain charge density (Coulomb explosion), which increases the surface area. This process is repeated until at the end either single ions are emitted from the remaining microdroplets (Ion Evaporation Model) or only single solvated ions are present in the droplets, which are completely desolvated by further drying (Charged Residue Model) [8-10]. The ions are transferred through a capillary or a small hole in the front plate via electric fields into the high vacuum of the mass spectrometer.
In atmospheric pressure chemical ionization, the analyte solution is sprayed through a heated (approx. 300 °C) ceramic tube and dried first. In an arc generated by a corona discharge needle, primary ionization of «air» takes place, which in this case is the reactant gas analogous to CI. In a second step, the charge is transferred from charged «air molecules» to the analyte molecules.
With ESI and APCI, the actual ionization takes place via protonation or deprotonation of the analyte molecules. Suitable analytes must therefore have protonable or deprotonable groups. The ionization process takes place outside the mass spectrometer. A comparison of the applications of the methods shows (cf. Fig. 2) that although APCI covers a lower molecular weight range, it is better suited for nonpolar compounds than ESI. Another important difference is that APCI produces single-charged analytes, whereas with ESI multiple charges may be present depending on molecular mass. As a rule of thumb it can be noted that with ESI on average one charge per 1000 mass units is transferred. This results in an almost unlimited ESI accessible molecular weight range.
Another ionization technique is Matrix Assisted Laser Desorption Ionization. The analyte molecules are mixed from solution with a matrix solution and crystallized on a “target”. This target is then transferred to the high vacuum of a mass spectrometer and irradiated with short laser pulses. The matrix absorbs the laser energy, evaporates and carries the embedded analyte molecules to the gas-phase. Ionization is performed by proton transfer between the matrix and the analyte molecules [11-14]. Due to the crystallization step, only indirect coupling with chromatography is possible, either manually or via corresponding robotics, which is hardly done today. Today, MALDI is mainly used for imaging applications in which a matrix-sprayed sample (e.g. tissue sections) is scanned two-dimensionally by the laser beam to obtain spatially resolved mass distributions that can be used to generate false color images [15,16].
ESI, APCI and MALDI are referred to as “soft” ionization techniques because there is no fragmentation due to ionization. ESI can also be used to generate mass spectra of non-covalent compounds. ESI and MALDI in particular are ideal for bioanalytics (proteins, peptides, etc.) due to their covered polarity and molecular weight range.
 
Other Ionization Techniques
In addition to the ionization techniques presented here in more detail, there are other techniques with low market penetration, such as Atmospheric Pressure Photo Ionization (APPI) or the relatively new SICRIT ion source, which are suitable for coupling separation techniques with mass spectrometers. For example, these two techniques can be used to couple gas and liquid chromatographs. It remains to be seen whether and to what extent these techniques will become more widely accepted in the future. 
 
The second part in the next issue will focus on HPLC-MS, CZE-MS and TLC-MS.
 
 
Authors
Uwe Linne1, Filipp Bezold1, Jan Bamberger1
Affiliation
1Department of Chemistry and Center for Synthetic Microbiology, Equipment Center Mass Spectrometry, Philipps-University Marburg, Marburg, Germany
 
Contact
Dr. Uwe Linne

Department of Chemistry and Center for Synthetic Microbiology
Core Facility of Mass Spectrometry
Philipps-University Marburg, Marburg, Germany

 

Virtual conference on MS in proteomics

 

Literature:
1] Gohlke R. S., McLafferty F. W. (1993), Early gas chromatography/mass spectrometry. J Am Soc Mass Spectrom. May;4(5):367-71. doi: 10.1016/1044-0305(93)85001-E.
2] Hübschmann, H.J. (1996), Handbook of GCMS - Fundamentals and Applications, Chemie Weinheim publishing house
[3] McLafferty F. W., Michnowicz J. A. Early gas chromatography/mass spectrometry, (1992) State-of-the-art GC/MS, Chemtech, American Chemical Society
[4] Beckey H. D. (1977) Principles of Field Desorption and Field Ionization Mass Spectrometry. Pergamon Press: Oxford
[5] Prokai L. (1990), Field Desorption Mass Spectrometry. Marcel Dekker: New York
6] Linden, H.B. (2004), Liquid injection field desorption ionization: a new tool for soft ionization of samples including air sensitive catalysts and non-polar hydrocarbons. Eur J Mass Spectrom (Chichester). 10(4):459-68.
[7] Yamashita M., Fenn J. B. (1984). Electrospray ion source. Another variation on the free-jet theme. The Journal of Physical Chemistry. 88 (20): 4451-4459. doi:10.1021/j150664a002
[8] Kebarle P., Verkerk U. H. (2009). Electrospray: from ions in solution to ions in the gas phase, what we know now. Mass Spectrom Rev. 28 (6): 898-917. doi:10.1002/mas.20247
[9] Nguyen S., Fenn J.B. (2007). Gas phase ions of solute species from charged droplets of solutions. Proc. Natl. Acad. Sci. USA. 104 (4): 1111-7. doi:10.1073/pnas.0609969104
 

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