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Mass Spectrometry Laboratory - Ionization Techniques

EI is the most commonly used method of ionization, and a great number of organic compounds are amenable to EI. To give an EI spectrum, the compound must be volatile and thermally stable. The sample may be solid, liquid or gas. Ions are formed when a 70 eV beam of electrons hits the sample molecules in the gas phase. This gives the sample molecules a great deal of excess energy and many fragment ions are formed. These ions can be useful in determining the structure of the molecule. Unfortunately, some compounds will fragment completely and not give molecular ions. Ionic samples generally do not work by EI. EI can be performed by direct probe and GC/MS. For compounds that do not work by EI, alternate methods of ionization have been developed.

CI is a relatively soft ionization technique. Thus CI produces less fragmentation than EI. CI can produce molecular ions for some volatile compounds that do not give molecular ions in EI. CI uses a reagent gas to gently transfer protons to the sample, usually producing (M+H)+ quasimolecular ions. We use both methane (CH4) and ammonia (NH3) on a routine basis for CI and when protonated in the ion source they form (CH5+ and NH4+) respectively. CI can be performed by direct probe and GC/MS.

Fast atom bombardment ionization (FAB or sometimes called liquid secondary ionization MS, LSIMS) is a softer ionization method than EI. It is one of the most popular ionization techniques for involatile and/or thermally labile molecules. FAB works best for polar and higher molecular weight compounds such as peptides and other biomolecules, ionic organometallics and other complex molecules. The commonly used matrices are glycerol, thioglycerol and 3-nitrobenzyl alcohol. The sample must be soluble in the matrix. A suitable type of matrix and sample are introduced on the sample target. Cs-gun generates a high energy beam of cesium ions which is directed on the sample target in the mass spectrometer ion source. The fast Cesium ions ionize the sample on the target by Cesium secondary ion MS (Cs-SIMS). FAB spectra are complex, showing (M+H)+ quasimolecular ions, fragments, and cluster groups of the matrix. If there are salts present in the sample, (M+Na)+ and/or (M+K)+ peaks will appear, and large amounts of salt will degrade the spectrum. In negative FAB spectra, the quasimolecular ion is usually (M-H)-.

The ESI source operating at atmospheric pressure. A solution of the sample is sprayed through a needle at potential of 3-6KV. Large charged droplets are produced by pneumatic nebulization. The droplet size gets smaller and the charge concentration at droplet's surface gets bigger when the solvent evaporates. Eventually, at the Rayleigh limit, coulombic repulsion overcomes the droplet's surface tension and the droplet explodes. This Coulombic explosion forms a series of smaller, lower charged droplets. This process is repeated until individually charged 'naked' analyte ions are formed. The charges are statistically distributed amongst the analyte's available charge sites, leading to the possible formation of multiply charged ions under the correct conditions. The most obvious feature of an ESI spectrum is that the ions carry multiple charges, which reduces their mass-to-charge ratio compared to singly charged species. This allows mass spectra to be obtained for large molecules. ESI is a soft and continuous ionization technique that is suitable for using as an interface with HPLC or Capillary Electrophoresis (CE). It is especially capable for large and/or labile molecules such as peptides, proteins, organometallics and polymers analysis.

In the Atmospheric Pressure Chemical Ionization mode, the analyte is delivered in a liquid stream from an LC system. When the liquid enters into a heated region of the APCI probe (up to 500 oC) it is desolvated and forced out of the probe in a nitrogen stream. At the exit there is a high voltage corona discharge needle, and the analyte is protonated by the H3O+ ions that are produced in the discharge region.

MS/MS is not an ionization technique but a method for structure determination and analysis of molecules. MS/MS employs two mass spectrometers in tandem. Between the two analyzers (MS1 and MS2) is a collision gas cell. Precursor ions selected by MS1 collide with a high pressure gas (usually He) in the cell and undergo fragmentation. The resulting daughter ions are analyzed by a B/E linked scan of MS2. The collision process is called collision induced dissociation (CID). MS/MS has proven useful in the sequence determination of peptides due to the formation of abundant daughter ions in the CID process. MS/MS can be run on molecular ions or fragment ions. MS/MS can also be performed with high resolution selection of the precursor for isobaric fragment species.

GCMS is used both for qualitative identification and quantitative measurement of individual components in volatile/semi-volatile organic complex mixtures. The GC effluent is directed into the mass spectrometer where the spectrum of each component is obtained as it elutes form the column. Electron Impact (EI) ionization is most commonly used to produce ions from the components. Chemical Ionization (CI) may also be used. Identification of the unknown organic compounds can be done by matching spectra with reference spectra and by spectral interpretation.

Although LC/MS is much more difficult than GC/MS because the liquid effluent is incompatible with the high vacuum of a mass spectrometer, it is still a popular technique for determining mixtures. We can perform ESI/APCI-LC/MS on a wide range of species. Buffers such as phosphate, tris, and hepes cannot be used. Only volatile buffers such as ammonium acetate can be used. Excess Na+, K+, and detergents are very bad for ESI/APCI and may result in no data.

CE is conducted in a small-diameter capillary that is under high potential (20-30KV). It is capable of ultrahigh resolution separations. The separation is based upon difference in the effective electrophoretic mobilities of analytes. The combination of CE with ESI-MS offers extraordinary resolving power and speed of analysis compared to conventional HPLC, while ESI-MS detection provides mass selectivity and structural information for compound identification. CE/MS has proven to be broadly applicable to a wide range of biologically important compounds.

The most sensitive way for measuring Dioxin in complex materials is High Resolution GC- High Resolution MS (HRGC-HRMS). The measurements are performed at a resolution power of 10,000. The 210 different isomers are measured by MID (Multiple Ion Detection) in five different fine windows. The stability and reliability of this method is ensured by measuring continuously a lock and calibration mass. The time window is related to the GC column. The program separates the dioxin by the difference of chlorination . With this kind of method it is possible to reach detection limits down to ppt.