Size exclusion chromatographic procedure

The validity of a practical method of direct viscosity calculation from size exclusion chromatographic analysis is demonstrated. The method is convenient to use and is not limited by the availability of narrow MWD standards. It is possible to accurately measure polymer Mark-Houwink constants using the suggested broad standard SEC-[n] and SEC-MW calibration procedure. 

This harmonious outcome from a simple procedure has far-reaching implications. That the species appearing at the void volume in aqueous 35% dioxane from Sephadex LH20 are associated is readily confirmed by the corresponding size-exclusion chromatographic profiles from a 106A pore-size... 

Principles and Characteristics Liquid chromatography is the generic name used to describe any chromatographic procedure in which the mobile phase is a liquid. It may be classified according to the mechanism of retention in adsorption, partition, size-exclusion, affinity and ion-exchange (Scheme 4.4). These mechanisms form the basis for the chromatographic modes of... 

A variety of procedures were utilized to analyze this reaction mixture and to characterize a,10-diaminopolystyrene. Thin layer chromatographic analysis using toluene as eluent exhibited three spots with Rf values of 0.85, 0.09, and 0.05 which corresponded to polystyrene, poly(styryl)amine and a,w-diaminopolystyrene (see Figure 1). Pure samples of each of these products were obtained by silica gel column Chromatography of the crude reaction mixture initially using toluene as eluent [for polystyrene and poly(styryl)amine] followed by a methanol/toluene mixture (5/100 v/v) for the diamine. Size-exclusion chromatography could not be used to characterize the diamine since no peak was observed for this material, apparently because of the complication of physical adsorption to the column packing material. Therefore, the dibenzoyl derivative (eq. 5) was prepared and used for most of the analytical characterizations. 

Figure 10.2 One of the procedures for entrapping an enzyme, E, (or any other solute) inside liposomes. The non-entrapped enzyme is eliminated by size exclusion chromatography, then the substrate (ADP in this case) is left to permeate throughout, the ADP excess is again eliminated chromatographically, and the reaction kinetics - due to the internalized enzyme reaction - measured. (From Walde et al., 1994b.)...

In the case of gel permeation or size-exclusion HPLC (HP-SEC), selectivity arises from differential migration of the biomolecules as they permeate by diffusion from the bulk mobile phase to within the pore chambers of the stationary phase. Ideally, the stationary phase in HP-SEC has been so prepared that the surface itself has no chemical interaction with the biosolutes, with the extent of retardation simply mediated by the physical nature of the pores, their connectivity, and their tortuosity. In this regard, HP-SEC contrasts with the other modes of HPLC, where the surfaces of the stationary phase have been deliberately modified by chemical procedures by (usually) low molecular weight compounds to enable selective retardation of the biosolutes by adsorptive processes. Ideally, the surface of an interactive HPLC sorbent enables separation to occur by only one retention process, i.e., the stationary phase functions as a monomodal sorbent. In practice with porous materials, this is rarely achieved with the consequence that most adsorption HPLC sorbents exhibit multimodal characteristics. The retention behavior and selectivity of the chromatographic system will thus depend on the nature and magnitude of the complex interplay of intermolecular forces... 

Procedure (See Chromatography, Appendix IIA.) Use a liquid chromatograph suitable for size-exclusion chromatography and equipped with a refractive index detector and a 60-cm x 7.5-mm (id) column packed with 5-p.m, 500A porosity PL-Gel, or equivalent, both operated at 40°. Operate the chromatograph at 500 to 1500 psi at a flow rate of 1.0 mL/min. 

This simple exercise demonstrates the chromatographic separation of the components of a mixture by a procedure that relies on two of the principles discussed above, namely ion-exchange chromatography and size-exclusion chromatography. The components to be separated (see Table 2-3) differ greatly from one another both in their molecular... 

Size-exclusion, or gel, chromatography is the newest of the liquid chromatographic procedures. It is a powerful technique that is particularly applicable to high-molecular-weight species."... 

Chromatographic patterns and data on the identity, homogeneity, and purity can be obtained by size exclusion chromatography, reverse-phase liquid chromatography, ion-exchange liquid chromatography, affinity chromatography, or other suitable procedures. 

However, fractional separation has been the basis for most asphalt composition analysis (Fig. 15.5). The separation methods that have been used divide asphalt into operationally defined fractions. Three types of asphalt separation procedures are now in use (a) chemical precipitation in which n-pentane separation of asphaltenes is followed by chemical precipitation of other fractions with sulfuric acid of increasing concentration (ASTM D-2006) (b) adsorption chromatography with a clay-gel procedure in which, after removal of the asphaltenes, the remaining constituents are separated by selective adsorption/desorption on an adsorbent (ASTM D-2007 and ASTM D-4124) and (c) size exclusion chromatography in which gel permeation chromatographic (GPC) separation of asphalt constituents occurs based on their associated sizes in dilute solutions (ASTM D-3593). 

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