Chromatography Data Acquisition Modes

No matter which ionization technique or chromatographic method is used, three acquisition modes exist scanning, selected-ion monitoring (SIM) (not to be mistaken with SIMS, which means secondary ion mass spectrometry) and selected-reaction monitoring (SRM). 

Chromatographic trace obtained by capillary electrophoresis and spectrum of the mixture of doubly or triply charged glycopeptides eluted together in the peak. Reproduced (modified) from Kelly J.F., Ramaley L. and Thibault P., Anal. Chem., 69, 51-60, 1997, with permission. 

The SRM acquisition mode allows one to obtain a sensitivity and selectivity gain with respect to SIM. The detection of selected reactions, based on the decomposition reactions of ions that are characteristic of the compounds to be analysed, requires the use of a tandem mass spectrometric instrument. In order to carry out this type of analysis, the spectrometer is set so as to let through only the ions produced by a decomposition reaction in the chosen reaction region for example, the first spectrometer selects the precursor ion with an mp+/z ratio that is characteristic of the compound to be detected, while the second spectrometer selects the fragment ion with an ntf+/z ratio resulting from the characteristic decomposition reaction of the compound to be analysed, mp 1 — ni(+ + mn, that occurs between the two analysers. 

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LC/MS/MS. LC/MS/MS is used for separation and quantitation of the metabolites. Using multiple reaction monitoring (MRM) in the negative ion electrospray ionization (ESI) mode, LC/MS/MS gives superior specificity and sensitivity to conventional liquid chromatography/mass spectrometry (LC/MS) techniques. The improved specificity eliminates interferences typically found in LC/MS or liquid chro-matography/ultraviolet (LC/UV) analyses. Data acquisition is accomplished with a data system that provides complete instmment control of the mass spectrometer. 

Pyrolysis-Gas Chromatography-Mass Spectrometry. In the experiments, about 2 mg of sample was pyrolyzed at 900°C in flowing helium using a Chemical Data System (CDS) Platinum Coil Pyrolysis Probe controlled by a CDS Model 122 Pyroprobe in normal mode. Products were separated on a 12 meter fused capillary column with a cross-linked poly (dimethylsilicone) stationary phase. The GC column was temperature programmed from -50 to 300°C. Individual compounds were identified with a Hewlett Packard (HP) Model 5995C low resolution quadruple GC/MS System. Data acquisition and reduction were performed on the HP 100 E-series computer running revision E RTE-6/VM software. 

Due to the limited time available for NMR data acquisition and the additionally reduced stability under flowing conditions, the on-flow mode is limited to the acquisition of ID spectra of the major peaks from a chromatographic separation. Minor compounds are normally not accessible. As no interruption or control of the chromatographic stage is necessary, the experiments can be carried out with standard chromatography equipment without the necessity of special equipment or software. The whole chromatogram is covered by the NMR spectra and all NMR-active compounds are detected. 

The basic setup for 1C is as follows. A pump is used to force the eluent through the system at a fixed rate, such as 1 mL/min. In the FILL mode a small sample loop (typically 10 to 100 pL) is filled with the analytical sample. At the same time, the eluent is pumped through the rest of the system, while by-passing the sample loop. In the INJECT mode a valve is turned so that the eluent sweeps the sample from the filled sample loop into the column. A detector cell of low dead volume is placed in the system just after the column. The detector is connected to a strip-chart recorder or a data-acquisition device so that a chromatogram of the separation (signal vs. time) can be plotted automatically. A conductivity- or UV-visible detector is most often used in ion chromatography. The hardware used in IC is described in more detail in Section 1.4. 

Gas chromatography/mass spectrometry (GC/ MS) of aromatic hydrocarbon fractions was done on an HP 6890 gas chromatograph equipped with an HP 7683 autosampler, a 60 m DB-1 column connected to an HP 5973 mass selective detector operated in the scan mode from 50 to 450 amu. On-column sample injection was done at programmed temperature at 3 °C above column oven temperature with constant flow helium carrier gas. The column oven programme initial temperature was 70 °C programmed to 315 °C (total run time 131.33 min). Internal standard (ortAo-terphenyl) at 300 ppm was used for quantification with data acquisition and integration on HP ChemStation software. Ratios may be found in Table 4. 

A very powerful combination results from the union of the high resolution capacity of gas chromatography with the identification capabilities of mass spectrometry. The resulting data have a tri-dimensional nature, from which retention times, chromatographic areas and mass spectra can be obtained for every single component of a complex mixture. The mass selective detector may operate in 3 acquisition modes (universal, selective, or specific) which facilitate the detailed characterization of complex mixtures such as those normally isolated... 

Metabolite ID 1.4 operates in both interactive and batch mode. In the interactive mode, the user reviews the full-scan data prior to MS/MS generation. In batch mode, the user submits a list of samples to be analyzed and starts automated acquisition. With such automated approaches, the metabolic profile of a single compound can be evaluated in approximately 1.5 hours, provided that adequate separation can be achieved with short, narrow-bore columns and fast-gradient chromatography. 

TOP analyzers are particularly useful when interfaced with chromatography where peak widths are measured in seconds it is important to obtain as much MS and MS/MS data as possible during that time period to fully characterize each individual chromatographic peak. In addition, the rapid acquisition of spectra at high resolution enables the collection of all data in accurate mass measuranent mode that can be further interrogated, e.g., to assist in the deconvolution of the components of overlapping LC peaks. 

QqQ in MRM mode and enables use of shorter chromatography columns and shorter run times (and thus increased throughput), while the additional selectivity provided by MRM detection can permit simplification of sample preparation procedures (Chapter 3). In practice, usually for each target analyte just one reaction channel (sometimes referred to as an MRM transition) is used to provide the quantitative data while one or two others are sometimes monitored simultaneously in order to provide confirmation of analyte identity via the relative responses (essentially a check on the selectivity of the analytical method. Section 9.4.3b). In contrast with the QqQ, the 3D ion trap has a poor duty cycle in MRM mode and the full scan product ion scan method is the method of choice if this analyzer is used for quantitation, since it is often possible to acquire 10 such scans across a chromatographic peak with adequate S/B values. Additional post-acquisition data processing is required to obtain quantitation data from such full scan MS/MS experiments. 

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