Ultrahigh-resolution

Figures 13.25-13.28 show the ultrahigh resolution separations in chloroform of polystyrene standards, polytetramethylene glycol, urethanes and isocyanates, and epoxy resins, respectively. Multiple column sets of anywhere from two to six columns in series have been used for well over a year with no apparent loss of efficiency. The 500- and 10 -A gels can easily tolerate 15,000 psi or more. In fact, the limiting factor in the number of columns that can be used in series is generally the pump or injector in the FIPLC system. A pump capable of 10,000 psi operation should allow the use of a column bank of 10-12 50-cm columns with a total plate count of 500,000 or more.

Katila et al. have carried out an unusual Mossbauer experiment of fundamental interest [65]. They have made use of the ultrahigh resolution of the 93.3 keV... 

Flughey, C. A. Hendrickson, C. L. Rodgers, R. R Marshall, A. G. Kendrick mass defect spectrum A compact visual analysis for ultrahigh-resolution broadband mass spectra. Anal. Chem. 2001, 73,4676-4681. 

Chan, A, Ko, TH, and Duker, JS, 2006. Ultrahigh-resolution optical coherence tomography of canthaxanthine retinal crystals. Ophthalmic Surg Lasers Imaging 37, 138-139. 

FIGURE 14.17 Ultrahigh resolution nano LC-MS separation of base peak chromatogram of 2351 peptides identified in trypsin digest of mouse brain lysate P2 fraction using Micro-Tech XtremeSimple nano-LC and Thermo Electron LTQ. Column 100 cm x 75 fim C18 column, 3 /mi, 8000 psi column head pressure. Solvent composition time 350 min gradient, 5 to 35% B. Solvent A 2% acetonitrile, 0.1% formic acid. Solvent B 95% acetonitrile, 0.1% formic acid. Data analysis Sequest, PeptideProphet, and Protein Prophet. 

The poor resolution of the fine structures of DNA and nucleosomes by conventional SEM was solved by the development of an ultrahigh-resolution SEM (14-15). Inaga et al. (16) observed the DNA double helix and nucleosomes of chicken erythrocytes by using an ultrahigh-resolution SEM. They modified the microspreading technique of Seki et al. (17) and combined it with the carbon plate method devised by Tanaka et al. (18). Briefly, they (procedures were performed at 0-4°C before fixation with the formalin solution) ... 

Viewed specimens with the ultrahigh-resolution SEM without any metal coating at accelerating voltages of 15 kV. 

Tohno et al. (20) used similar steps to determine the chromatin loop size in human leukemia cells (an ultrahigh-resolution EM was not needed). To determine chromatin loop size, the lengths of 102 chromatin fibers protruding from four nuclei were measured and their average lengths were estimated. The steps they used (essentially those of Miller and Bakken (21) are summarized as follows ... 

Inaga S, Osatake H, Tanaka, K. SEM images of DNA double hehx and nucleosomes observed by ultrahigh-resolution scanning electron microscopy. J Electron Microsc 1991 40 181-186. 

Tanaka K, Mitsushima A, Yamagata N, Kashima Y, Takayama H. Direct visualization of colloidal gold-bound molecules and a cell surface receptor by ultrahigh resolution scanning electron microscopy. J Microsc 1990 161 455—461. 

Example Peptides often contain sulfur from cysteine. Provided there are at least two cysteines in the peptide molecule, the sulfur can be incorporated as thiol group (SH, reduced) or sulfur bridge (S-S, oxidized). Often, both forms are contained in the same sample. At ultrahigh-resolution, the contributions of these compositions to the same nominal m/z can be distinguished. The ultrahigh-resolution matrix-assisted laser desorption/ionization (MALDI) FT-ICR mass spectrum of native and reduced [D-Pen jenkephalin gives an example of such a separation (Fig. 3.25). [39] The left expanded view shows fully resolved peaks due to and C2 isotopomers of the native and the all- C peak of the reduced compound at m/z 648. The right expansion reveals the peak of the native plus the... 

Fig. 3.25. Ultrahigh-resolution MALDI-FT-ICR mass spectrum of native (S-S) and reduced (2 X SH) [D-Pen ]enkephalin. The expanded m/z views of the second and third isotopic peak show fully mass-resolved signals. Reproduced from Ref. [39] with permission. American Chemical Society, 1997.

Solouki, T. Emmet, M.R. Guan, S. Marshall, A.G. Detection, Number, and Sequence Location of Sulfur-Containing Amino Acids and Disulfide Bridges in Peptides by Ultrahigh-Resolution MALDI-FTICR Mass Spectrometry. Anal. Chem. 1997,69, 1163-1168. 

Marshall, A.G. Detection, Number, and Sequence Location of Sulfur-Containing Amino Acids and Disulfide Bridges in Peptides by Ultrahigh-Resolution... 

High and in particular ultrahigh-resolution in combination with a soft ionization method such as ESI, MALDI, or FD presents another way to achieve the separation of the molecular species contained in a mixture. Given a sufficient level of resolution, isobaric ions are displayed separately in the range of their common nominal mass value (Chap. 3.3.2, 3.4). 

Classically, high-resolution work is the domain of double-focusing magnetic sector instruments. More recently, TOP and to a certain degree triple quadrupole instruments are also capable of resolutions up to about 20,000. However, the rapid development of FT-ICR instruments has established those as the systems of choice if ultrahigh-resolution (>100,000) and highest mass accuracy (1 ppm) are required (Chap. 4.6). 

The potential of ultrahigh-resolution mass spectrometry for the analysis of complex chemical mixtures is particularly illustrated by FT-ICR-MS which definitely sets a new standard. For example, ultrahigh-resolution was applied to separate several thousand components in crude oil, [85,86] fuels, [87,88] or explosion residues. [89]... 

The ruffled heme appears to result from llel23 and Ilel33 pushing down from the distal pocket side (3.4 and 3.5 A), and from His59, the proximal histidine, pushing up from below (3.4 and 3.5 A), Fig. 17. Despite the ultrahigh resolution of the structure, residues 31-37 are sufficiently disordered that side-chain positions cannot be modeled. 

G. R. Asbury and H. H. Hill, Jr., Evaluation of Ultrahigh Resolution Ion Mobility Spectrometry as an Analytical Separation Device in Chromatographic Terms, J. Microcolumn Sep. 2000,12, 172 H. E. Revercomb and E. A. Mason, Theory of Plasma Chromatography/Gaseous Electrophoresis, Anal. Chem 1975,47,970. 

L.G. Randall, Ultrahigh Resolution Chromatography. A.Ahuja (editor), ACS Symposium Series, 1984. 

Columns vary in physical size depending on the job to be accomplished and the packing material used. There are four basic column sizes microbore (l-2mm i.d.), analytical (4-4.5mm i.d.), semipreparative (10-25mm i.d.), and preparative (1-5 in i.d.). Column lengths will range from a 3-cm ultrahigh resolution, 1 -3-pm packed microbore column to a 160-cm semipreparative column with 5 pm packing. The typical analytical column is a 4.2-mm i.d. x 25-cm C18 column packed with 5 pm media. 

Sleighter, R. L., and Hatcher, P. G. (2007). The application of electrospray ionisation coupled to ultrahigh resolution mass spectrometry for the molecular characterization of natural organic matter. I. Mass Spectrom. 42, 559-574. 

Stenson, A. C., Landing, W. M., Marshall, A. G., and Cooper, W. T. (2003). Exact masses and chemical formulas of individual Suwannee River fulvic acids from ultrahigh resolution electrospray ionisation fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 75,1275-1284. 

Hockaday, W. C., Grannas, A. M., Kim, S., and Hatcher, P. G. (2006). Direct molecular evidence for the degradation and mobility of black carbon in soils from ultrahigh-resolution mass spectral analysis of dissolved organic matter from a fire-impacted forest soil. Org. Geochem. 37, 501-510. 

Kim, S., Kramer, R. W., and Hatcher, P. G. (2003). Graphical method for analysis of ultrahigh-resolution broadband mass spectra of natural organic matter, the Van Krevelen diagram. Anal. Chem. 75, 5336-5344. 

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