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Analysis Methods

Low resolution EI/CI (Electron Ionization/Chemical Ionization) - GCT instrument

In the ideal case, the EI spectrum of a sample compound will provide the molecular weight (as the m/z value of the [M].+ ion), in addition to the corresponding m/z values of the fragment ions. Fragment ion peaks may provide structural information about the sample compound analyzed. As a result of the high energy of the ionization process (the sample is bombarded by 70 eV electrons), the molecular ion may not be observed. Note that unknown sample compounds can sometimes be identified by searching a database. The NIST library, which contains spectra for approximately 150,000 compounds, is commonly used for database searches.
Chemical ionization (CI) is a much softer ionization process and so increases the probability of generating and observing the molecular ion. In this process, a reagent gas (called the CI gas - usually NH3) is introduced into the ion source. Bombarding electrons interact mainly with the CI gas molecules which become ionized. Upon introduction into the source, the sample molecules are ionized indirectly by ion-molecule reactions with the ionized CI gas.  Ions observed are usually species such as [M+H]+ and [M+NH4]+. Note that this is not always the case, for example, if your sample has a proton affinity significantly lower than NH3, you may still see [M]
.+ or even [M-H]+ instead of [M+H]+.  Often, it is preferable to run both EI and CI on the same sample to provide more confidence in the assignment of the molecular ion.


High resolution EI/CI - GCT instrument

An accurate mass measurement on a compound can be useful for confirming a proposed molecular formula, particularly if for some reason the elemental composition cannot be measured.  The measured mass, with some assumed accuracy (generally ± 5 ppm), is submitted into a program which returns the possible molecular formulae. Usually constraints are included on the number of possible atoms to limit the number of retrieved formulae.
Note that determination of a molecular formula from an accurate mass measurement alone is a chancy business, since the accuracy required for unambiguous formula determination increases rapidly as molecular mass increases. For example, (see the Journal of the American Society for Mass Spectrometry instructions to authors), with the formula constrained to C0-100 , H3-74, O0-4, N0-4, only ~30 ppm accuracy is required for unambiguous identification at a mass of 118. At a mass of 500, 5 ppm accuracy allows for 5 possible formulae. When the mass is increased to 750, with an accuracy of 5 ppm and a formula constraint of C0-100 , H25-110, O0-15, N0-15, 626 possibilities are allowed and an accuracy of better than 0.02 ppm would be required. Thus it is clear that for masses above 500 Da, an accuracy beyond 5 ppm is required, in fact, as much accuracy as possible is ideal to limit the possible retrieved formulae using MS data. The use of other information such as fragmentation patterns, intensity of isotopic peaks, chemistry of the system in question, can be critical.


GC/MS

GC/MS measurements are performed on the GCT instrument interfaced to a Agilent 6890 Series GC System. The Agilent 6890 has a J&W DB-XLB 30m column and an autosampler. This is particularly useful in analysis of mixtures - samples may contain either several analytes of interest or impurities which interfere with MS analysis of the compound of interest.

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GC trace of a pesticide mixture

 

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MS of each component of pesticide mixture and their respective NIST reference spectra.

 



Low resolution ESI/APCI (Electrospray Ionization/Atmospheric Pressure Chemical Ionization) - Quattro Ultima instrument

The ESI or APCI spectrum of your compound will almost always give an unambiguous molecular weight. Both processes of ion formation (ESI/APCI) are low energy and result in relatively little fragmentation. The ion observed is usually not precisely the 'molecular ion'. In positive ion mode, species such as [M+H]+, [M+NH4]+, [M+Na]+ and [M+K]+ are common and in negative ion mode, species such as [M-H]-, [M+Cl]- and [M+HC(=O)O]- may be observed. The possibility of multiple ion attachment in ESI allows mass measurement of very high molecular weight species, such as proteins. For example, the attachment of 30-40 protons to a protein of MW 40 kDa gives signals in the m/z range 500-2000, which can be easily measured. These multiply charged ion signals are deconvoluted by a program (Maximum Entropy) included in the software. This deconvolution process generates a zero-charge protein spectrum.

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Raw data of Mut AroA.

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Deconvoluted spectrum of Mut AroA using the Maximum Entropy calculation. (P.Berti)


Observation of cation or anion radicals ([M].+, [M].-) is rare in ESI, but common in APCI. ESI in particular requires relatively little material - in normal operating procedures, 10 uL of  solution in the mM-nM concentration range are injected whereas 'nanospray' operation requires even less.
Structural information on a particular ion may be obtained by MS/MS. The ion is mass selected by the first quadrupole and transmitted into the second quadrupole where it is collisionally fragmented. The fragment ions are mass analyzed using the third quadrupole and subsequently detected to obtain an MS/MS (or CID) spectrum. This spectrum can be used for structure elucidation and is sometimes called the 'fingerprint' spectrum.


LC/MS

LC/MS and LC/MS/MS measurements are carried out on the Quattro Ultima instrument, which is interfaced to a Waters 2690 High Performance Liquid Chromatograph (HPLC). The Waters 2690 has a ternary pump system, column compartment temperature control and an autosampler. This is particularly useful in analysis of mixtures - samples may contain either several analytes of interest or impurities which interfere with MS analysis of the compound of interest.


 

High resolution ESI (Electrospray Ionization) - Global Ultima instrument

High resolution ESI data is similar to that obtained on the low resolution instrument, but offers higher sensitivity, resolution and mass accuracy.

 


Protein/Peptide Identification and Sequencing - Global Ultima instrument

MSMS sequencing of a tryptic peptide of Cytochrome c.


 

MALDI (Matrix-Assisted Laser Desorption/Ionization)- Micromass TofSpec 2E.

Compounds are observed as [M]+, [M+H]+, [M+Na]+, etc., in positive ion mode, and [M]- or [M-H]- in negative ion mode.  This is a relatively gentle ionization mode, but usually more fragmentation is observed than in ESI.  Complications can arise because of fragmentation and interactions with matrix-derived moieties.

Material requirements are quite low, for example, for peptides1 ul of a 1 uM solution is sufficient.

MALDI Spectrum of PNIPAM (poly-N-isopropylacrylamide) polymer. (Stover group)