Sample Fractionation

Although on-line HPLC-MS using an electrospray interface is a straightforward technique nowadays, it can be of advantage to analyze specific samples off-line. 

In the case of MALDl-MS, the nanoHPLC has to be coupled to the mass spectrometer offline. After separation, the sample is transferred to a sample plate (target), e.g. AnchorChip target (Broker Daltonics, Bremen, Germany), equipped with a special coating that provides improved crystallization of M ALDI samples and a 10- to 100-fold sensitivity increase. Small hydrophilic positions/anchors (600 pm diameter) are applied equidistantly on the hydrophobic surface of the AnchorChip target thereby reducing sample spreading to only a small area. 

This results in co-crystaUization of matrix and sample and therefore higher sample concentration focused at the distinct hydrophilic anchor points [16]. One of the most commonly used matrices is a saturated solution of a-cyano-4-hydroxy-cinnamic acid in 97% acetone/3% water containing a small amoimt of TFA to support the ionization of the sample molecules in the MALDI source. The matrix is directed to the AnchorChip target and this is then dried. The sample fractionation after HPLC can be performed automatically using special sample fractionation systems (e.g., Probot, LC Packings Dionex). The target is introduced to a special desk and can be adjusted in the three XYZ planes so that the small nano-LC volume is placed directly onto the matrix. Using this set-up, fractions of volume less than 40 nL can be collected automatically. After collection, the individual fractions are recrystallized from 0.01% TFA, 30% acetone, and 60% ethanol in water and analyzed. 

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Both preparative and analytical GPC were employed to analyze a standard (NBS 706) polystyrene sample. Fractions were collected from the preparative column, the solvent was evaporated away, and the weight of each polymer fraction was obtained. The molecular weights of each fraction were obtained usmg an analytical gel permeation chromatograph calibrated in terms of both and M. The following data were obtained ... 

FIGURE 4.49 Isolation of a complex protein conjugate on Toyopearl HW-50S. Column 22 mm X 83 cm. Sample Fraction from crude Tetrahymena H2A containing the ubiquitin-histone conjugate uH2A. Elution 10 nM HCI. Flow rate 0.1 ml/min. Detection UV at 230 nm. 

Figure 12.8 Mia ocolumn size exclusion chromatogram of a styrene-aaylonitrile copolymer sample fractions ti ansfeired to the pyrolysis system are indicated 1-6. Conditions fused-silica column (50 cm X 250 p.m i.d.) packed with Zorbax PSM-1000 (7p.m 4f) eluent, THF flow rate, 2.0 p.L/min detector, Jasco Uvidec V at 220 nm injection size, 20 nL. Reprinted from Analytical Chemistry, 61, H. J. Cortes et al, Multidimensional chromatography using on-line microcolumn liquid chromatography and pyrolysis gas chromatography for polymer characterization , pp. 961 -965, copyright 1989, with peimission from the American Chemical Society.

Conventional SEC calculations were performed with the SEC software package GPCl (Chromatix). As described below a universal calibration was carried out to obtain the peak position molecular weight at each V a modified version of GPCl then was used to calculate weight-and number-average molecular weights for the sample fraction at V (Appendix). 

Because of chromatographic dispersion, the sample fraction in the detector cell is polydisperse. The weight-average and number-average molecular weights of the polydisperse fraction are calculated as... 

Multidimensional and Multimodal Sample Fractionation and Trace Enrichment Techniques.790... 

Sampling by incorrect (non-representative) treatment of individual sample fractions. 

The ability to resolve and characterize complicated protein mixtures by the combination of 2DLC and online mass spectrometry permits the combination of sample fractionation/simplification, top-down protein mass information, and bottom-up peptide level studies. In our lab, the simplified fractions generated by 2D(IEX-RP)LC are digested and analyzed using common peptide-level analysis approaches, including peptide mass fingerprinting (Henzel et al., 1993 Mann et al., 1993), matrix-assisted laser desorption/ionization (MALDI) QTOF MS/MS (Millea et al., 2006), and various capillary LC/MS/MS methodologies (e.g., Ducret et al., 1998). 

This is an analytical technique used for the separation and identification of additives. It can also be used in a preparative way for the isolation of sample fractions for subsequent chromatographic or spectroscopic analysis. Thiocarbanilide... 

One approach that allows increased chromatographic flow rates without loss of resolution entails the use of microparticulate stationary-phase media of very narrow diameter. This effectively reduces the time required for molecules to diffuse in and out of the porous particles. Any reduction in particle diameter dramatically increases the pressure required to maintain a given flow rate. Such high flow rates may be achieved by utilizing high-pressure liquid chromatographic systems. By employing such methods, sample fractionation times may be reduced from hours to minutes. 

A total of 112 samples of rock rose (roots and leaves) and 56 soil samples (fraction <0.18 mm) where these plants were grown were analyzed. Soil pH was determined in a soil/water suspension with 1/2.5 (m/v) proportion. Soils and plants were analyzed for Cu, Zn Al, Fe, Mn, Ca, K and P by ICP-OES after digestion with four acids (HCIO4+HNO3+HCI+HF). 

In addition to MALDI-TOF and LC-MS/MS, SELDI-TOF-MS can also be used to determine expression profiling of various biological samples, such as serum or plasma for early detection of infection. Serum proteomic profiling assay, for instance, has been used to distinguish patients with acute SARS (severe acute respiratory syndrome) from patients with fever and influenza with 100% accuracy [16]. A major limitation of SELDI-TOF-MS, however, is that it cannot be used for direct amino acid sequence identification of the biomarker proteins, necessitating further sample fractionation and protein purification. 

In Figure 5, we show some lineshapes obtained from some of our samples at 77 K. Each point represents the size of a spin echo obtained at that field. The dispersion of these samples (fraction of Pt atoms which are on the surface of the particles) were 4, 11, 15, 26, 46, and 58% for Figures 5a through 5f, respectively. These samples were untreated, i.e., exposed to air. The width of most of these lines is about 4 kG. Compared to Hi = 100 G, these lines are indeed broad ... 

During the development of sample-fractionation methods, the main goal is to achieve the highest resolution possible between the molecule of interest and the contaminants. Fractionation by SEC is used both as an analytical tool and as a... 

Corrections for instrumentally-produced mass fractionation that preserve natural mass dependent fractionation can be approached in one of two ways a double-spike method, which allows for rigorous calculation of instrumental mass fractionation (e.g., Dodson 1963 Compston and Oversby 1969 Eugster et al. 1969 Gale 1970 Hamelin et al. 1985 Galer 1999 see section Double-spike analysis ), or an empirical adjustment, based on comparison with isotopic analysis of standards (Dixon et al. 1993 Taylor et al. 1992 1993). The empirical approach assumes that standards and samples fractionate to the same degree during isotopic analysis, requiring carefully controlled analysis conditions. Such approaches are commonly used for Pb isotope work. However, it is important to stress that the precision and accuracy of isotope ratios determined on unknown samples may be very difficult to evaluate because each filament load in a TIMS analysis is different. 

The right hand-side of these two equations is simply the slope of the isotopic ln(r,) vs. ln(r ) array for a same sample fractionated to different extents. For instance, we obtain the expression ofthe slope of the alignment of Fe isotopic ratios of a same sample in the... 

Surface enhanced laser desorption/ionization (SELDI) is a distinctive form of laser desorption ionization where the target plays an active role in the sample preparation procedure and ionization process [49]. Depending on the chemical or biochemical treatment, the SELDI surface acts as solid phase extraction or an affinity probe. Chromatographic surface is used for sample fractionation and purification of biological samples prior to direct analysis by laser desorption/ ionization. SELDI is mainly applied for protein profiling and in biomarker discovery by comparing protein profiles from control and patient groups. 

The instruments for polymer HPLC except for the columns (Section 16.8.1) and for some detectors are in principle the same as for the HPLC of small molecules. Due to sensitivity of particular detectors to the pressure variations (Section 16.9.1) the pumping systems should be equipped with the efficient dampeners to suppress the rest pulsation of pressure and flow rate of mobile phase. In most methods of polymer HPLC, and especially in SEC, the retention volume of sample (fraction) is the parameter of the same importance as the sample concentration. The conventional volumeters— siphons, drop counters, heat pulse counters—do not exhibit necessary robustness and precision [270]. Therefore the timescale is utilized and the eluent flow rate has to be very constant even when rather viscous samples are introduced into column. The problems with the constant eluent flow rate may be caused by the poor resettability of some pumping systems. Therefore, it is advisable to carefully check the actual flow rate after each restarting of instrument and in the course of the long-time experiments. A continuous operation— 24h a day and 7 days a week—is advisable for the high-precision SEC measurements. THE or other eluent is continuously distilled and recycled. 

Compound Formula Whole Sub sample fraction Mutagenic potency c relative to BaP = 1.00 1 Whole Sub sample fraction... 

No sample fractionation procedure is given in the protocol, but several possible techniques were included in the literature review, including acid/base-neutral extraction, solvent fractionation (8), column chromatography, TLC, and HPLC (presented elsewhere in this chapter). 

The concentrated extract is then split. To one portion, 1.0 mL of DMSO is added, and the dichloromethane is removed under a stream of nitrogen. The resulting DMSO solution is used for the direct assay of the extract. If fractionation of the remaining extract is required, the investigator is given the option of using the acid/base-neutral extraction scheme described in step 3 of the nonaqueous liquid protocol or the HPLC technique described in the sample fractionation methods section. 

Sample Fractionation Methods. There was a consensus among panel participants that each of the protocols shared areas where further... 

Figure 2. A large particle from sample fraction KH-3. The particle is spherical with many spheres stuck to its surface...

Chemical Composition Aerosol composition measurements have most frequently been made with little or no size resolution, most often by analysis of filter samples of the aggregate aerosol. Sample fractionation into coarse and fine fractions is achieved with a variety of dichotomous samplers. These instruments spread the collected sample over a relatively large area on a filter that can be analyzed directly or after extraction Time resolution is determined by the sample flow rate and the detection limits of the analytical techniques, but sampling times less than 1 h are rarely used even when the analytical techniques would permit them. These longer times are the result of experiment design rather than feasibility. Measurements of the distribution of chemical composition with respect to particle size have, until recently, been limited to particles larger than a few tenths of a micrometer in diameter and relatively low time resolution. One of the primary tools for composition-size distribution measurements is the cascade impactor. 

The results of the stability studies showed that AOZ concentrations in the bound and extractable sample fractions of liver decreased by 22% and 27%, respectively, at +4°C after 48 h. However, liver samples that had been frozen at — 20°C showed no significant difference in AOZ concentration after 6 months of storage (148). Therefore, AOZ seems to be a good marker residue for bound residues of FZD in tissue samples (149). 

Sampling Fractionation Research Inc. (FRI) developed a sam-ling technique that eliminates the influence of "end effects and etects a maldistributed composition profile. This technique [Silvey and Keller, IChemE Symp. Ser. 32, p. 4 18 (1969)] samples the bed at frequent intervals, typically every 0.6 m or so. HETP is determined from a plot of these interbed samples rather than from the top and bottom compositions. 

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