Size exclusion chromatogram

In a report, Hellwig and coworkers attempted to standardize particular types of caramel by means of the following tests (a) with citric acid, (b) solubility in 65% (v ) ethanol, and (c) the Lassaigne test (see Table 11). They also presented gel-permeation (g. p.) chromatograms (size exclusion chromatography) of various types of caramel. Values of for zones on the chromatograms evidently characteristic for the caramels studied are attributable to color components of caramel. These studies were directed to caramels standardized by ITCA. They allow division of these caramels into ten types and into further subtypes (see Table III). 

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). 

Size-exclusion chromatography can be carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram. 

An example of a size-exclusion chromatogram is given in Figure 7 for both a bench-scale (23.5 mL column) separation and a large-scale (86,000 mL column) mn. The stationary phase is Sepharose CL-6B, a cross-linked agarose with a nominal molecular weight range of 5000-2 x 10 (see Fig. 6) (31). 

Fig. 7. Chromatograms of size-exclusion separation of IgM (mol wt = 800,000) from albumin (69,000) where A—D correspond to IgM aggregates, IgM, monomer units, and albumin, respectively, using (a) FPLC Superose 6 in a 1 x 30 — cm long column, and (b) Sepharose CL-6B in a 37-cm column.

Fig. 14. Molecular weight characteristics of novolac resins. Shown is the size-exclusion chromatogram for a typical commercial novolac polymer. The unsymmetrical peak shape reflects the multimodal molecular weight distribution of the polymer.

Other lignins show different polydispersity as demonstrated by high pressure size exclusion chromatograms (47). The polydispersity of lignosulfates is much greater, with M ratios in the range of 6—8 (48). 

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.

Size exclusion chromatography (SEC) separates molecules of a polymer sample on the basis of hydrodynamic volume. When the chromatograph is equipped only with a concentration-sensitive detector, i.e. conventional SEC, a molecular weight distribution (MWD) can be obtained from the chromatogram only through use of a calibration function relating molecular weight and elution volume V (2). 

Figure 4.20 Size-exclusion chromatogram of SBR adhesive formulation in THF using UV and RI detectors. After Alfredson and Tschida [760]. Reproduced by permission of Varian, Inc.

Figure 4.21 High-performance size-exclusion chromatograms of the polymeric plasticiser Reoplex R346 using (a) RI and (b) UV detection. After Castle et al. [792]. Reproduced from L. Castle et al., Food Addit. Contain., 8, 565-576 (1991), by permission of Taylor Francis Ltd (http //www.tandf.co.uk/journals)...

Figure 2. Size exclusion chromatograms of polystyrene standard (A) and p(t-butyl styrene)-b-PSX (B).

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. 

One column can be used for different types of liquid chromatography by changing the eluent components. As an example, a column packed with octadecyl-bonded silica gel has been used for size-exclusion liquid chromatography with tetrahydrofuran (THF), normal-phase liquid chromatography with n-hexane, and reversed-phase liquid chromatography with aqueous acetonitrile. Examples of the chromatograms are shown in Figure 1.4. 

On the basis of these preliminary experimental results we are confident the proposed model is capeible of explaining many of the experimentally observed features of size exclusion chromatograms. It is perhaps appropriate to comment further on the physical importance of the major pareuneters of interest in the model. 

Size exclusion chromatography (SEC) polymer elution profiles yield information regarding the molecular size distributions of polydisperse macromolecules. Polymer molecular weight distribution (MWD) represents an intrinsic property which provides direct correlation with many end-use physical properties and a universal criterion for polymer characterization (1). In order to convert elution profiles or chromatograms into MWD information proper calibration methods are required. SEC molecular weight calibration techniques represent experimental approaches for transformation of polymer elution profiles into MWD information and are dependent upon instrumentation, columns, and the polymer/solvent system under study. 

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