Size exclusion chromatogram analysis

Examples of the application of size-exclusion chromatography to the analysis of proteins. The separation in (a) uses a single column that in (b) uses three columns, providing a wider range of size selectivity. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

Molecular Weight Classes Used for the Analysis of the Size Exclusion HPLC Chromatograms of Gelatin... 

Two different sample preparation methods by size-exclusion and reverse-phase chromatography were proposed for analysis of PCs and PAs in wine. In the former, a volume of 5 mL of dealcoholized wine is passed through a Fractogel Toyopearl TSK gel HW-50 (F) (12 x 120 mm) column. The stationary phase is washed with 25 mL of water and the simple polyphenols are eluted with 50 mL of an ethanol/water/trifluo-roacetic acid 55 45 0.005 (v/v/v) solution. The polymeric fraction is recovered with 50mL of acetone/H20 60 40 (v/v). Figure 6.19 shows the LC/ESI-MS extracted ion chromatograms of dimers and trimers in a wine (Fulcrand et al., 1999). 

The selection of the particle size of a column involves several compromises. One is the compromise between analysis time, resolution, and backpressure discussed in general in Chapter 3. The second is the selection of the range in the chromatogram in which we desire maximum resolution and the conditions associated with this. The third is the compromise between the desire to maximize resolution and the prevention of breakdown of the sample due to shear degradation. The second and third are specific to size-exclusion chromatography. 

The analysis of a broad-polydispersity polymer (PD > 1.2) can be carried out by combining GPC or size-exclusion chromatography (SEC) with MALDI-MS [161-172]. In this approach, a wide-polydispersity polymer is first separated by GPC and fractions at a defined time interval are collected. The time interval is properly chosen so that the individual fraction would contain only a narrow-polydispersity (PD < 1.2) polymer, which can then be analyzed by MALDI-MS for accurate molecular mass determination. The molecular mass information generated from the MALDl analysis of aU individual fractions can be used to convert the time domain in the GPC chromatogram into a mass domain. The polymer distribution can be determined from this chromatogram. 

Different liquid chromatography modes in polymer analysis were successfully interfaced with electrospray ionisation time-of-flight mass spectrometry in a single experimental set-up the mass spectrometry data from size exclusion chromatography/mass spectrometry of PMMA were used as absolute calibration points in the size exclusion chromatography/refractive index chromatogram, and monomer mass and end groups were inferred from the isotopically resolved mass spectra. 44 refs. 

LCCC is a powerful tool for the analysis of block copolymers. Lee et al. analyzed a copolymer containing blocks of poly(ethylene oxide) and blocks of poly(lactide) by LCCC, and tiiey were able to determine the conditions in which chains with lactic blocks of different size are eluted at almost the same time. The LCCC chromatogram shows a series of narrow peaks, and the MALDI-TOF spectra of the LCCC fractions confirm that each structure contains exclusively one type of oligomer. 

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