Chromatographic timescale

Udeshi, N.D. Shabanowitz, J. Hunt, D.F. Rose, K.L. Analysis of proteins and peptides on a chromatographic timescale by electron-transfer dissociation MS. FEES J. 2007, 274, 6269-6276. 

Fourier transform (FT) analyzers. The FT instruments have the highest available resolution. Currently there are two types, the orbitraps and the ion cyclotron resonance (ICR) systans. Mass resolution in orbitraps can reach 250,000, while ICR systems can have resolutions of >3,000,000. In these analyzers ions oscillate/rotate within a cell and are detected by recording the electrical current that the passage of ions induces in the snrfaces of the cell. The resolution attainable is inversely proportional to both the mass of the analyte ion and the time required to acquire the data. A consequence of this proportionality is that the highest resolutions cannot be obtained on the chromatographic timescale where mnltiple spectra must be collected to enable the characterization of peaks that are only a few seconds wide. LIT/FT combinations are the most common form of MS/MS systems that ntilize orbitrap and ICR analyzers (Sections 2.3.4 and 23122). 

A difficulty with both the orbitrap and ICRMS is that their ability to provide very high resolution depends on keeping the ions in the analyzer for considerable periods of time, and this may not be compatible with the chromatographic timescale. For instance, when analyzing an ion of miz 800 with a 9.8 Tesla magnet, a 5 s transient is required to attain a resolution of -500,000, while a 0.5 s transient provides a... 

TOF analyzers are the fastest instruments for the collection of high-resolution spectra. The current generation of TOF analyzers is capable of collecting mnltiple spectra on a chromatographic timescale, where peaks are only a few seconds wide, at resolutions of 20,000-40,000 (or even 60,000) with accurate mass measurement. On the other hand, there is a progressive reduction of the resolution attainable on FT ion traps as the speed of the production of spectra is increased (Section 2.3.4.2). This has led to an alternative MS/MS strategy a portion of the molecular ion signal is passed... 

Holland JF, Enke CG, Allison J, et al. (1983) Mass spectrometry on the chromatographic timescale Realistic expectations. Analytical Chemistry 55(9) 997A-1012A. 

FIGURE 11.1 Split peak (1.380-1.436) of interconverting free and paired analytes if the rate of the process is slow compared to chromatographic retention timescale. 

In view of the short timescale of the analytical cycle, step changes in the concentrations of species in the stream flowing through the mini-column, and the low pressure and temperature involved, several chemical species can be simultaneously retained and eluted, i.e. the process involves a non-chromatographic separation [205]. These are the main differences between flow analysis exploiting SPE and liquid chromatography. 

The third situation is when the objectives (usually specific and limited in scope) require the highest resolution (>1,000,000) that is available only with FT-ICRMS systems. Because the timescales of data acquisition for these instruments are not compatible with LC, the current approach is to bypass the chromatographic step and use flow injection to introduce samples, and then use an LIT (often included) in conjunction with the resolving power of the FT-ICR to tease apart highly complex mixtures (e.g., top-down proteomics or crude oils). It is likely that the organization contemplating such an instrument already has other mass spectrometers along with experienced operators/researchers. 

Thus far the only difference between the UV and El detectors is that the observed signal for the former is a measure of transmitted intensity I, while that for the latter is a measure of the absorbed intensity — but it turns out that is of little consequence for the present purpose. Thus, for example, a fluorescence detector records a signal that is a measure of the absorbed intensity of the exciting radiation like the El case, but it is also a concentration dependent detector like the simple UV absorption detector. The difference between the two types arises rather in the relationship between the analyte concentration delivered by the mobile phase (c ) and that within the absorption cell or El source in the former case c = c (see equation [4.2]), but the situation is very different in the El case where the mass spectrometer vacuum pumps continuously remove the analyte from the El source. In fact c ei represents an instantaneous steady state value, a compromise between the flow rate of A into the source and the pumping rate out of the source here instantaneous means simply that the establishment of the steady state value c gi occurs on a timescale appreciably shorter than that of the chromatographic peak. Then at this steady state ... 

A major barrier to development of MALDl as an off-line interface between HPLC and MS for trace quantitation has been the chemical noise (peak-at-every-m/z background, particularly at low mh <500) where matrix-derived peaks can dominate the spectrum. However, the overall problem can be resolved into constituent parts, namely, development of efficient deposition of eluant to provide adequate representations of the chromatographic separation, and efficient MALDl-MS analysis of the deposited analytes on a timescale that should certainly be no longer than the chromatographic time, and also methods to provide acceptable accuracy, precision, repeatability, LOD, LLOQ and dynamic range of the quantitation. 

Another recent approach has been to insert an ion mobility device between the ion source and mass spectrometer. The additional separation power added by the differences in ion transit times through a buffer gas can often distinguish between analyte ions and the background ions that originate in the ion source (and thus can not be chromatographically separated). This multidimensional approach is feasible because of the differences in timescales among the HPLC peaks (seconds), ion mobility (tens of milliseconds) and SIR or MRM dwell times (a few... 

Analysis of DCLs by a combination of high-performance liquid chromatography (HPLC) and MS (LC-MS) has proven very powerful, provided the library members are stable enough to allow chromatographic separation. LC-MS analysis can yield both the quantity and the identity of individual library members. Care should be taken that the exchange between library members is slow on the timescale of the experiment. This is usually the case for hydrazone and disulfide libraries, without requiring special precautions. It has recently been reported that even the composition of DCLs of hydrolytically labile imines can be analyzed directly by HPLC, provided an acidic mobile phase is used. Beau et al. successfully used a common reversed-phase HPLC solvent system based on acetonitrile/water mixtures containing 0.1% trifluoroacetic acid [10]. Imine hydrolysis is slow under these conditions because at low pH the zwitterionic hemi-aminal intermediate that is believed to be important in the hydrolysis process is more difficult to attain (Scheme 2.1). 

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