Size exclusion experiments

Porous silica packings do, however, sometimes suffer from adsorption between the sample and silanol groups on the silica surface. This interaction can interfere with the size exclusion experiment and yield erroneous information. In many cases, this problem is easily overcome by selecting mobile phases that eliminate these interactions. In addition, the surface of porous silica packings is routinely modified in order to reduce these undesirable interactions. Trimeth-ylsilane modified packing is typically used with synthetic polymers. Diol modified packing is typically used with proteins and peptides. 

Thus by using Co2(C0)8 as a representative organometallic guest, and a combination of size exclusion experiments and intrazeolite oxidation chemistry, as probed through the far-IR cation spectra of sodium and acid faujasites and A-zeolites, one is able to distinguish between internal and external confinement of the metal guest(s) in the zeolite. 

This mechanism is supported by results from gel filtration experiments with the phospho-mimetic mutant Ser463Asp of a coronin 3 (1C) C-terminal peptide, as well as the fuU-lei th protein, which elute as a dimeric species. The wild type peptide and protein, in contrast, elute as a trimer in the size exclusion experiments. Also, phosphorylation at Ser463 regulates the oligomerisation state of coronin 3 (1C) and subsequently alters its cellular activity. 

Supramolecular chemists without BLM-workstation can approximate the inner diameter of synthetic ion channels and pores by size exclusion experiments in LUVs. Differences in activity between the HPTS assay - compatible with all diameters - and the ANTS/DPX assay reserved for pores with diameter larger than 5 A and the CF assay for pores with larger than 10A can differentiate between ion channels and pores (Fig. 11.5). Larger fluorescent probes like CF-dextrans are available to identify giant pores or defects [26]. However, we caution... 

Parallel and antiparallel self-assembly can be differentiated by voltage dependence Parallel self-assembly of asymmetric monomers gives voltage-sensitive, antiparallel self-assembly voltage-insensitive synthetic ion channels and pores [9]. Other indirect evidence from function such as inner diameters from Hill analysis of single-channel conductance (Section 11.3.3) or other size exclusion experiments is often used to support indications for supramolecular active structures molecular modeling can be of help as well [3, 4, 10]. 

High performance liquid chromatography (HPLC). All HPLC analyses were performed with a model HP1090 analytical HPLC equipped with a diode array detector (Hewlett Packard, Mountain, View, CA). A Protein PAK 3000 SW column (Waters, Bedford, MA) was used for each size exclusion experiment. The column was equilibrated with ten column volumes (100 ml) of elution buffer (2.0 M GuHCl, 50 mM Tris sulfate, 5 mM EDTA, pH 7.5) prior to operation. A sample volume of 25 gl was applied to the column and eluted at a flow rate of 1.0 ml/min to facilitate rapid separation. For equilibrium experiments, each sample was equilibrated for three to eight hours before column separation. 

Fig. 1. Chromatograms illustrating difference between a small zone (a) and a large zone (b) size exclusion experiment. In the saull zone experiment, the elution volume, V, is taken as the apex of the peak In a large zone experiment, the elutxon volume is the centroid volume, V, of the leading boundary. The shaded areas represent the volume loaded onto the column in each case.

A wealth of information can be gleaned from a large zone size exclusion experiment. By studying the behavior of the leading boundary over a range of concentrations, one can immediately ascertain whether a rapid equilibrium exists under the conditions being studied. If there is an equilibrium, the stoichiosietry of the self-association can be deduced, and finally the equilibrium constaut can be determined. 

Conductivity detectors, commonly employed in ion chromatography, can be used to determine ionic materials at levels of parts per million (ppm) or parts per bUHon (ppb) in aqueous mobile phases. The infrared (ir) detector is one that may be used in either nonselective or selective detection. Its most common use has been as a detector in size-exclusion chromatography, although it is not limited to sec. The detector is limited to use in systems in which the mobile phase is transparent to the ir wavelength being monitored. It is possible to obtain complete spectra, much as in some gc-ir experiments, if the flow is not very high or can be stopped momentarily. 

This chapter makes no distinction between gel-permeation chromatography (GPC) and size exclusion chromatography (SEC). We make mention of specific analysis conditions wherever possible. We have attempted to include a variety of conditions but by no means should this chapter be considered a comprehensive review of conditions for analyzing polyacrylates. We have drawn extensively from our own experience in selecting examples. 

Ghijs, M., DeWaele, C., and Sandra, R, Experiments with size exclusion material in microchromatography. Part 1. Microcolumn SEC, ]. HRC, 13, 651, 1990. 

This presentation demonstrates that a small minicomputer can be used to provide a full range of functions for collection and interactive reduction of data from a size-exclusion liquid chromatograph. A number of different users have collected in excess of 5000 chromatograms using this equipment. The experience gained with this system has influenced our approach to the automation of other analytical instruments. Careful attention to control paths, provision of "user friendly" access to the system functions, and careful management of the data archiving functions are crucial to the success of such efforts. 

Many of the possible column combinations that are useful in 2DLC are listed in Chapter 5. Besides the actual types of column stationary phases, for example, anion-exchange chromatography (AEC), size exclusion chromatography (SEC), and RPLC, many other column variables must be determined for the successful operation of a 2DLC instrument. The attributes that comprise the basic 2DLC experiment are listed in Table 6.1. 

Size-exclusion chromatography (SEC) has been used to characterize the unimer-micelle distribution. However, SEC is not an absolute method and thus requires calibration. Since it is practically impossible to calibrate a SEC apparatus for the unimers and micelles formed by a block copolymer, only indicative MW values can be obtained. Moreover, several authors have noted a strong perturbation of the unimer-micelle equilibrium during SEC experiments even when interaction of the material with the SEC column was minimized [4,61,62],... 

Buchacher et al. [43] discussed the continuous separation of protein polymers from monomers by continuous annular size exclusion chromatography. The P-CAC used for the experiments was a laboratory P-CAC type 3 as described in Table 1. The results were compared to conventional batch column chromatography in regard to resolution, recovery, fouling, and productivity. The protein used in the studies was an IgG preparation rich in aggregates. Under the conditions used, the polymers could be separated from the monomers, although no baseline separation could be achieved in either the continuous or the batch mode. The... 

In addition, the determination of a polymer s endgroup(s) [125,126] and the analysis of random and block-copolymers [127,128] can be achieved by MALDI. However, care has to be taken when judging the MALDI spectra because of the mass-dependent desorption and detection characteristics of the experiment. In case of higher polydispersity (PD >1.1) high-mass ions are underestimated from MALDI spectra. [93,124] The current practice to deal with such samples is to fractionate them by gel permeation chromatography (GPC) [123] or size-exclusion chromatography (SEC) prior to MALDI analysis. [124,129]... 

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