Gel-exclusion

Kessel D, Thompson P (1987) Purification and analysis of hematoporphyrin and hematoporphyrin derivative by gel exclusion and reverse-phase chromatography. Photochem Photobiol 46 1023-1025. 

We foimd that the ratiometric method is superior because it is not dependent on pyranine concentration and therefore free of error in pipeting (18,22,54). Calibration curves were constructed by preparing liposomes in which the hydration of the lipids to form MLV was done using solutions of high concentration at the desired pH in the range of 3.0 to 10.0. Gel-exclusion chromatography on a Sephadex column, as mentioned above, yielded a series of liposome preparations with a fixed external pH (pH 7.5), but different internal pH values determined by the buffer used for lipid hydration. Neither KI nor DPX, which quench the fluorescence of aqueous solutions of pyranine, has much effect on the fluorescence intensity of pyranine in the void volume after gel-exclusion chromatography, which indicates the complete removal of the pyranine from the extraliposomal medium. 

Y = ratio between [ C]-methylamine counts (dpm) and phospholipid concentration (mM) in the void volume fraction after the separation by gel-exclusion chromatography. Percentage of encapsulation (%) = Y/Xx 100. 

Figure 2 (right). Reverse-phase HPLC elution profile of the tumor-localizing fraction of HPD isolated by non-aqueous gel exclusion chromatography. (B) Hydrolysis of this material in 50% aqueous THF containing IM HCl, at 37 °C for 24 hours. (C) After hydrolysis in IM NaOH, 50% aqueous THF under the same condition. The major porphyrins resulted are hematoporphyrin (HP), hydroxyvinyl deutero-porphyrin (HVD), and protoporphyrin (PP). 

Partial Purification of SA-GTase. Proteins extracted from oat roots Incubated for 20 h in salicylic acid were separated by salt precipitation and gel exclusion and anion exchange chromatography (Table IV). A 5 -fold purification of the SA-GTase was achieved with this... 

Hofsten and Lalasidis (15), however, reported that plasteins subjected to gel exclusion chromatography in 50X acetic acid showed no increase in molecular size over that of the reactants. Monti and dost (29) reached the same conclusion based on gel chromatography in DMSO and on analysis of a-amino nitrogen in plasteins. Hofsten and Lalasidis (15) noted that hydrophobic peptides showed unusual elution behavior on sephadex gels in water or dilute buffers, providing a possible explanation for differences in their results compared to those of Arai et al. 

Pallavicini et al. (16) utilized a-chymotrypsin immobilized on chitin to catalyze plastein formation from leaf protein hydrolyzates. When analyzed by gel exclusion chromatography, the products were comparable to those produced by soluble enzymes. Modification of Specific Functional Properties... 

The great versatility of HPLC is evidenced by the fact that all chromatographic modes, including partition, adsorption, ion exchange, chromato-focusing, and gel exclusion, are possible. In a sense, HPLC can be considered as automated liquid chromatography. The theory of each of these chromatographic modes has been discussed and needs no modification for application to HPLC. However, there are unique theoretical and practical characteristics of HPLC that should be introduced. 

The combination of HPLC and gel exclusion chromatography is used extensively for the separation of large biomolecules, especially proteins and nucleic acids. 

Selection of type of chromatography (partition, adsorption, ion exchange, gel exclusion). 

Selection of the matrix used to immobilize a ligand requires consideration of several properties. The stationary supports used in gel exclusion chromatography are found to be quite suitable for affinity chromatography because (1) they are physically and chemically stable under most experimental conditions, (2) they are relatively free of nonspecific adsorption effects, (3) they have satisfactory flow characteristics, (4) they are available with very large pore sizes, and (5) they have reactive functional groups to which an appropriate ligand may be attached. 

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