Size exclusion chromatography retention volume

Fig. 5.1. Diagrammatic illustration of the parameters involved in size exclusion chromatography. Fq, volume of solvent outside the particles V, volume of solvent inside the particles available for chromatography, (A), elution volume of a sample A (B), elution volume of a sample B. From this, the retention coefficient can be derived for each sample ...

ISEC is a size-exclusion chromatography technique, in which the stationary phase is the CFP to be to characterized [16-18] and the eluates are geometrically well-defined steric probes. From the determined retention volumes in a given solvent and on the basis of suitable morphological models, ISEC analysis provides the... 

Size exclusion chromatography (SEC), also known as gel permeation chromatography (GPC), was used for the separation and fractionation of macromolecules on an analytical and preparative scale [17]. The separation occurs predominantly by the hydrodynamic volume of the macromolecules in solution, however, in some cases the polarity of the molecules can also influence the retention times. Like HPLC, the SEC technique is generally very reproducible with regard to its elution times (typically < 1 h) and hence can be used for automated synthesis. But because the cost for an automated SEC system is high, it must be considered as a serial separation technique. In addition, larger scale separations > 100 mg, usually require repetitive injection of small aliquots. 

Calibration of size-exclusion chromatography columns based on the finding that the retention volume of a molecular or particulate species is usually a single-valued function of an appropriate size parameter of this molecule or particle, irrespective of its chemical nature and structure. 

Characteristic of the efficiency of a size-exclusion chromatography set-up in terms of band broadening, defined as N = (y la Y, where Fr is the retention volume of an individual low-molecular-weight compound, and oy is the corresponding full width at 60.7% peak height of the elution peak. 

The specific pore volume is important in size-exclusion chromatography (SEC) [10] because the separation takes place there. In retentive chromatography, it is necessary to provide the surface area... 

The separation of chemical species by size exclusion chromatography is more reproducible than any other type of chromatography. Once the SEC columns, the mobile phase (most often a pure solvent like THF or toluene), and the flow rate are selected, the retention volume (or retention time assuming the flow rate does not change) is primarily a function of linear molecular size, which can be obtained from the valence bond structure if the compound is known. Some of the chemical species can interact with the solvent forming complexes with an effective linear size greater than that of the molecule. This causes the expected retention volume, based on "free" molecular structure, to shift to a lower but very reproducible retention volume. Phenols in coal liquids form 1 1 complex with THF (9,10) and carry the effective linear molecular size to increase. As a result phenolic species elute sooner than expected from their... 

The role of size exclusion chromatography is the separation of rather complex coal liquids into simpler fractions. The retention volume can be used to help Identify the chemical structure where GC-MS is unable to identify its possible structure. For example biphenyl and dihydroacenaphthene have the same molecular formula as well as similar mass spectral fragmentation patterns. Coal liquids contain both species. The one which appears first (lower SEC retention volume) is biphenyl (GC ret. time = 17 min. in Figure 5-6). Dihydroacenaphthene appears later at longer SEC retention volume and is identified in Figure 5-12 at GC retention time of 13 minutes. The former has a longer structure compared to the latter. 

Dependence of Spreading Factor on the Retention Volume of Size Exclusion Chromatography... 

FIG. 1.23 Plot of log M and detector output versus retention volume for size-exclusion chromatography. Also shown is the relation among VR, Vv, VP, and K, Vp as discussed in the text. (Redrawn with permission of P. C. Hiemenz, Polymer Chemistry The Basic Concepts, Marcel Dekker, New York, NY, 1984.)... 

Ball, D. Thornton, T. "Advantages of Direct Measurement of Retention Volume in Liquid Chromatography and Size Exclusion Chromatography" 32nd Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, NJ, 1981 paper 357. 

The effect of limited penetration of the pores by the largest molecules may also be applied beneficially for the separation of very large molecules. Depending on the size of the molecules (in solution), they will be more ore less excluded from the pores, and hence the retention times will be affected. This effect is used in size exclusion chromatography (SEC) or gel permeation chromatography (GPC). In this technique, any interactions between the solute molecules and the stationary phase are purposefully avoided. The solute molecules remain exclusively in the mobile phase, but the accessible mobile phase volume, and hence the retention volume, may vary between the total volume of the mobile phase and the so-called exclusion volume, which is the total volume of mobile phase outside the pores. The latter elution volume applies to very large solute molecules (excluded solutes),... 

Since size exclusion chromatography separates polymer molecules by their size (especially hydrodynamic size), plotting the molecular size vs. the retention volume should be universal, regardless of the polymer molecular weight. The universal calibration curve is given as ... 

In Fl-FFF, the channel is created by placing a mylar spacer with the channel cut out between two porous frits. A membrane hlter of a specihc molecular weight cutoff is placed on one of the frits and acts as the accumulation wall to permit flow, without loss of particles. The applied force is then a perpendicular flow of the carrier solution across the porous frits. Fl-FFF is a versatile technique capable of separating macromolecules as small as roughly 1000 Da, in which case it is comparable to gel permeation (size exclusion) chromatography. However, Fl-FFF can also be applied to the separation of colloidal particles. In this case the hydrodynamic diameter of the colloidal particle is related to the retention volume, V by the equation... 

Determination of the average chemical composition and polymer composition by size-exclusion chromatography (SEC) has been reported in the literature. Two different types of concentration detector or two different absorption wavelengths of an ultraviolet or an infrared detectors are employed the composition at each retention volume is calculated by measuring peak responses at the identical retention points of the two chromatograms. 

In order to calculate the molecular weight M) or molecular-weight distribution (MWD) of the polymer, the dependence of the Soret coefficient on M must be known. Because is virtually independent of M, at least for random coil polymers, the dependence of retention on M reduces to the dependence of D on M. The separation of molecular-weight components by D (or hydrodynamic volume, which scales directly with D) is a feature that thermal FFF shares with size-exclusion chromatography (SEC). In the latter technique, the dependence of retention on D forms the basis for universal calibration, as D scales directly with the product [rjjM, where [17] is the intrinsic viscosity. Thus, a single calibration plot prepared in terms of log([i7]M) versus retention volume (F,) can be used to measure M for different polymer compositions, provided an independent measure of [17] is available. In thermal FFF, a single calibration plot can only be used for multiple polymers when the values of for each polymer-solvent system of interest are known. However, a single calibration plot can be used with multiple channels. In... 

Tanaka and Ishizuka [3] studied the separation of acids by ion-exclusion chromatography on a modern, high performance cation-exchange column. Hitachi cation exchange resin, 8 % cross-linking, 18 2 pm particle size was used. Retention volumes of acids were obtained at 50 °C using water as the eluent. These are given in Table 8.1. 

Differentiation and separation only occurs over a certain range of molecular sizes, typically between molecular weights of 2 kDa and 200 kDa, although this can be increased up to 1,000 kDa by the use of more specialised gels. This size range is dependent on the sizes of the pores and pore size distribution in the gel matrix. Retention volumes Vr are often used in size exclusion chromatography instead of retention times tR. The total volume V, of the separation column is the sum of the volume of the gel particles Vg, the volume of the solvent inside the pores, also called the intrinsical volume V and the volume of the free solvent outside the pores, the inter particle volume Vq ... 

The retention factor in size-exclusion chromatography is defined relative to the elution volume of an excluded peak ... 

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