Molecular exclusion properties

In view of the large number of new zeolites recently synthesized, considerable effort has been expended in their physical characterization, in particular, via their sorption capacities for various organic substrates. The molecular exclusion properties of these zeolites have been used to estimate their pore-opening catacteristics and shape-selective properties (6). In contrast to the molecular sieving... 

Attempts have been made to incorporate into toothpastes enzymes such as dextranase and mutanase (a-1 6- and a-1 3-glucosidase respectively) which degrade plaque polysaccharides. These enzymes work well in laboratory tests, but seem to be unable to penetrate plaque in situ due to the molecular-exclusion properties of the plaque matrix (page 497). 

Alhedai et al also examined the exclusion properties of a reversed phase material The stationary phase chosen was a Cg hydrocarbon bonded to the silica, and the mobile phase chosen was 2-octane. As the solutes, solvent and stationary phase were all dispersive (hydrophobic in character) and both the stationary phase and the mobile phase contained Cg interacting moieties, the solute would experience the same interactions in both phases. Thus, any differential retention would be solely due to exclusion and not due to molecular interactions. This could be confirmed by carrying out the experiments at two different temperatures. If any interactive mechanism was present that caused retention, then different retention volumes would be obtained for the same solute at different temperatures. Solutes ranging from n-hexane to n hexatriacontane were chromatographed at 30°C and 50°C respectively. The results obtained are shown in Figure 8. 

The exact nature of the dead volume is complex and, in fact, will vary from solute to solute due to the exclusion properties of the stationary phase, particularly if the stationary phase or support is silica or silica based. Thus, to measure (Vo) accurately, a non-adsorbed solute of the same molecular size as the solute should be used and then the correct retention volume (V r) can be calculated and employed for identification purposes. 

The factors that control separation and dispersion are quite different. The relative separation of two solutes is solely dependent on the nature and magnitude of the Interactions between each solute and the two phases. Thus, the relative movement of each solute band would appear to be Independent of column dimensions or particle geometry and be determined only by the choice of the stationary phase and the mobile phase. However, there is a caveat to this statement. It assumes that any exclusion properties of the stationary phase are not included in the term particle geometry. The pore size of the packing material can control retention directly and exclusively, as in exclusion chromatography or, indirectly, by controlling the access of the solute to the stationary phase in normal and reverse phase chromatography. As all stationary phases based on silica gel exhibit some exclusion properties, the ideal situation where the selective retention of two solutes is solely controlled by phase interactions is rarely met in practice. If the molecular size of the solutes differ, then the exclusion properties of the silica gel will always play some part in solute retention. 

Some of the characteristic column volumes outlined in the previous argument were determined by Alhedal et al (11) in the examination of a commercially available reverse phase column packing, Zorbax Cs. These authors examined the exclusion properties of the interstitial volume of the column by measuring the retention volume of a number of salts of different molecular volume.The substances used, in ascending order of ion volume, were as follows,... 

It is seen that an approximately linear relationship exists between the retention volume of each alkane and Its carbon number and that the smaller molecule exhibits the greatest retention. This is a direct result of the exclusion properties of the silica gel support. The fact that the data, taken at the two different temperatures, fall on the same straight line confirms that little or no partition is taking place and that the difference In retention between the Individual solutes is entirely related to their molecular size. 

It follows that retention measurements on silica based stationary phases for the purpose of obtaining thermodynamic data is fraught with difficulties. Data from solutes of different molecular size cannot be compared or related to other Interacting variables ideally, thermodynamic measurements should be made on columns that contain stationary phases that exhibit no exclusion properties. However, the only column system that might meet this requirement is the capillary column which, unfortunately introduces other complications wmcn will be discussed later. 

At the earlier conferences on molecular sieves, in London in 1967 and in Worcester, Mass. (U.S.) in 1970, attention was focused exclusively on the zeolites. In an etymological sense (separation of molecules according to size by selective diffusion through pores of appropriate diameter) the field of molecular sieves must not be restricted to the tectosilicates with porous framework. This point is developed by R. M. Barrer in Chapter 1, where he gives a broad review of those compounds which can exhibit molecular sieve properties. 

Molecular exclusion matrix with properties suitable for purification of the specific conjugate Sephadex G-200 (Pharmacia Biotech, Uppsala, Sweden), B10-Gel A-0.5 m or Bio-Gel A-l 5 m (Bio-Rad Laboratories, Hercules, CA) are useful for relatively small to large conjugates, respectively... 

Inspection of Eq. 7 reveals that the molecular interference function, s(x), can be derived from the ratio of the total cross-section to the fitted IAM function, when the first square bracketed factor has been accounted for. A widely used model of the liquid state assumes that the molecules in liquids and amorphous materials may be described by a hard-sphere (HS) radial distribution function (RDF). This correctly predicts the exclusion property of the intermolecular force at intermolecular separations below some critical dimension, identified with the sphere diameter in the HS model. The packing fraction, 17, is proportional for a monatomic species to the bulk density, p. The variation of r(x) on 17 is reproduced in Fig. 14, taken from the work of Pavlyukhin [29],... 

Employ sequential separation processes based on different physical, chemical, or biochemical properties that are synergistic, and thus orthogonal, rather than repetitive, i.e., use a metal ion-affinity HPLC procedure prior to a size-exclusion step, a hydrophobic interaction HPLC step after an ion-exchange HPLC procedure, a biomimetic HPLC step before a biospecific affinity HPLC step, etc. This rule is in accord with the orthogonality rule, which anticipates that the most efficient separation procedures are ones that take advantage of the anisotropy of molecular physicochemical properties of the target protein or polypeptide rather than the commonality of the molecular features. 

The laboratory of C. L. Hew has more recently described the composition and structure (C. L. Hew, personal communication), the physical properties (Ananthanarayanan and Hew, 1977a), and the biosynthesis (Hew and Yip, 1976) of the antifreeze protein from P. americanus. Molecular exclusion through Sephadex G-75 was used to identify the numbers and sizes of the antifreeze proteins in the... 

The bottom line conclusion of this Section can be stated thus If two analytes A and B are to be chromato-graphically separated, their adjusted retention volumes V, and V,g (Equation [3.15]), and thus the corresponding retention times tj, must be sufficiently different. To separate two solutes, either their distribution coefficients and K (Equation [3.1]) must be made to differ (choose appropriate phase systems) or the volumes of stationary phase with which they interact must be made to differ (choose a stationary phase with appropriate exclusion properties based on molecular size so that the effective values of are different for different analytes) or a shrewd combination of both. It is appropriate to mention that if mass spectrometric detection is used, clean chromatographic separation of solutes in the analytical extract is not as crucial as for less selective detectors in view of the additional selectivity provided by the m/z information. 

Equation (36) is certainly more accurate than equation (34) but still does not take into account any exclusion properties that the support may have. In addition, the particles are close-packed and touching so, in the interstitial volume, around the points of contact, some additional solute exclusion will almost certainly take place. The pore size of most silicas can range from 1-3 A to several thousand angstrom and so can easily partially exclude some of the components of a mixture that cover a wide range of molecular size. It follows that equation (36) must be further modified. 

The regular windows and channels in zeolites with specific dimensions are the basis for their shape-selective properties and accordingly their utiUzation in purification of gas mixtures or mixtures of branched and linear hydrocarbons. The ability to preferentially adsorb certain molecules and simultaneously excluding others led to the introduction of the term molecular sieves for zeoUtes and their appUca-tions in separation techniques based on their size-exclusion properties. 

Other than the above structural features there are two important and exclusive properties that make DNA suitable for molecular level constructions. These are molecular recognition and self-assembly. The nucleotide bases A and T on two different ss-DNA have affinity towards each other, so do G and C. Effective and stable cis-DNA structures are only formed if the base orders of the individual strands are complementary. Hence, if two complementary single strands of DNA are in a solution, they will eventually recognize each other and hybridize or zip-up forming a cis-DNA. This property of molecular recognition and self-assembly has been exploited in a number of ways to build complex molecular structures [ 114-121]. In the mechanical perspective, if the free energy released by hybridization of two complementary DNA strands is used to lift a hypothetical load, a force capacity of 15 pN can be achieved [122], comparable to that of other molecular machines like kinesin (5 pN) [123],... 

In some respects it is misleading to refer to pores in SEC media, since this often implies the existence of well defined spaces in a matrix composed of stationary elements. At the molecular level, the gel forming elements of many of the most useful media have a mobility not much different from the mobihty they would have in free solution. Any individual space in such a gel is continually changing both its size and shape, just those properties which determine its ability to exclude other molecules by steric exclusion. The dynamic nature of the spaces in these gels means that the pore size distribution must be defined operationally in terms of its exclusion properties [9]. It almost certainly explains the observation that pore size distributions for gels where the exclusion properties are defined by flexible polymer chains are smoother than for those for gels where the excluding elements are expected to be stiffen... 

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