Mass Spectrometry Basics

A sample is analyzed in a mass spectrometer by ionizing the sample and separating ions of differing masses and recording their relative abundance by measuring intensities of ion flux. A typical mass spectrometer comprises three parts: an ion source, a mass analyzer, and a detector.

The sample to be analyzed is ionized in the ion source. The ions are then transported by magnetic or electrical fields to the mass analyzer. The mass analyzer separates the ions according to their mass to charge ratio (m/z). There are many types of mass analyzers. Many mass spectrometers use two or more mass analyzers for tandem mass spectrometry (MS/MS). The final element of the mass spectrometer is the detector. The detector records the charge induced or the current produced when an ion passes by or hits a surface.

The use of a mass spectrometer as the detector in gas chromatography or HPLC combines the features of gas or liquid chromatography and mass spectrometry to identify different substances within a sample. In GC/MS or LC/MS, a gas or liquid chromatograph is used to separate compounds chromatographically before they are introduced to the ion source and mass spectrometer.

Use the information below as a guide to determine which ionization method is best suited for your sample.

Electron Ionization (EI)

Electron Impact (EI) is the original ionization used in mass spectrometry. The sample is vaporized into the mass spectrometer ion source, where a beam of electrons with sufficient energy (typically 70 eV) will ionize the molecules present.

EI is useful for structural characterization, as an EI mass spectrum usually contains the molecular ion, and many fragment ions. Some samples will fragment completely and not give molecular ions. The presence of a molecular ion in a spectrum generally decreases with increasing saturation, particularly if heteroatoms are present.

EI is best suited for the analysis of relatively non-polar, volatile samples. Analytes have to be vaporized therefore is restricted to thermally stable compounds with low molecular masses. Ionic samples generally do not work by EI.

Chemical Ionization (CI)

CI is a soft ionization technique. In CI a reagent gas (e.g. methane (softest), isobutane, or ammonia (hardest)) is first ionized by electron impact. Sample ions are then formed by the interaction of these reagent ions and sample molecules. Ion molecule reactions occur and pseudo-molecular ion [M+H]+(positive ion mode) or [M-H]- (negative ion mode) are formed. Such ions have very little tendency to fragment because they are even electron species and little excess energy is imparted to them.

It is a useful technique when no molecular ion is observed in EI. CI is more appropriate than EI for more polar compounds.

Electrospray Ionization (ESI)

ESI is a soft ionization method producing fewer in-source fragment ions. In ESI, a sample solution is passed through a metal capillary biased at high potential (4-5 kV) to form droplets. The droplets carry charge when the exit the capillary and as the solvent vaporizes the droplets disappear leaving charged analyte molecules. An ESI mass spectrum usually consists of a series of multiply-charged ions for large molecules such as proteins.

ESI works well for small or large molecules. It is suitable for analyzing polar, even ionic compounds and large bio- or synthetic polymers.

Buffers such as phosphate, tris, and hepes cannot be used. Excess Na+, K+, and detergents habitually result in no data. Detergents (PEGs and PPGs) are especially bad because they are ionized very well by ESI and suppress ionization of analytes of interest.

Atmospheric-pressure chemical-ionization (APCI)

APCI is one of the mildest ionization techniques available. A sample solution flows through a heated tube where it is volatilized and sprayed into a corona discharge to create a plasma. In this plasma proton transfer reactions and some fragmentation can occur.

APCI is best suited to relatively polar, semi-volatile samples. It is suitable for analyzing less polar compounds compared to ESI, although fragmentation is increased compared to ESI.

Matrix-assisted laser desorption ionization (MALDI)

The sample is mixed with a suitable MALDI matrix and allowed to co-crystallize on a metal surface. A laser beam is then directed onto this surface, causing desorption and ionization of sample components by a combination of desorption and ionization processes. A MALDI mass spectrum usually consists of singly charged ions for each sample component.

The efficiency of MALDI has been shown to be highly sensitive to the sample preparation procedure. Many variables influence MALDI sample, including concentration of the matrix and analyte, choice of matrix, contaminants and compatible solubilities of matrix and analyte solutions, analyte sample history (i.e., exposure to ionic detergents).

It is a soft ionization method producing less fragmentation. MALDI is the method of choice for large and/or labile molecules such as peptides, proteins, and polymers.

Low levels of some salts, buffers, and detergents can be tolerated as well as less than 2% of glycerol. However, data quality and sensitivity may be compromised. Water, acetonitrile, methanol, THF, and other volatile organic solvents can be used. DMSO and DMF cannot be used.