Chromatographic void volume

Porosity is one of the most important properties of a stationary phase, since it severely inflnences the chromatographic colnmn performance, the speed of separation, as well as the specific surface area and consequently loading capacity. Porosity refers to the degree and distribution of the pore space present in a material [114], Open pores indicate cavities or channels, located on the surface of a particle, whereas closed pores are situated inside the material. The sum of those pores is defined as intraparticular porosity. Interparticular porosity, in contrast, is the sum of all void volume between the particles. According to their diameter, pores have been internationally (lUPAC) classified as follows [114] ... 

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... 

Sulfosalicylic acid has most commonly been used to precipitate proteins prior to ion-exchange amino acid analysis (11). In this mode, SSA allows for a very simple sample preparation that requires only centrifugation of the precipitated sample and then direct injection of the resulting supernatant solution. The supernatant solution is already at an appropriate pH for direct injection. Also, the SSA does not interfere chromatographically since it elutes essentially in the void volume of the column. It has been noted that, if an excessive amount of SSA is employed, resolution of the serine/threonine critical pair can suffer (12). The use of SSA prior to reversed-phase HPLC can be more problematic, since its presence can interfere with precolumn deriva-tization. For example, Cohen and Strydom (13) recommend the separation of the amino acids from the SSA solution on a cation-exchange resin prior to derivatization with phenylisothiocya-nate (PITC). 

A problem with the chromatographic determination of cysteic acid is that there is almost no retention of cysteic acid. For both reversed-phase HPLC and ion-exchange amino acid analyzers (usually employing cation-exchange resins), cysteic acid is essentially eluted within or near the void volume of the column. This makes it more susceptible to unknown chromatographic interferences from various matrices. When cysteine is alkylated by 3-bromopropylamine, the product (S-3-aminopropylcysteine) looks very similar to lysine in structure. Hale et al. (90) show that this alkylated species affords excellent chromatographic separation on four different commercially available amino acid analysis systems and that, indeed, it does elute very near lysine in each case (see Fig. 4). 

The volume of the NMR detection cell is relatively large (30-240 xl) when compared with the peak volumes and other void volumes in the chromatographic system. This leads to a considerable broadening of the peaks when they pass the NMR detection cell. It takes a long time until a peak is completely washed out of the flow cell, i.e. a tailing is observed. This is especially critical when traces of a high-concentration first peak interferes with the spectrum of a minor compound. 

Zinc-binding components of urine were examined using modified gel chromatography (15). Urine (3 ml) was chromatographed on Sephadex G-25 columns (2.5 x 40 cm) equilibrated with a buffer containing 10 ppm Zn as Zn(N03)2 and 10 mM Tris buffer, pH 7.4. Fractions of 3 ml were analyzed by atomic absorption spectroscopy. Void volume of the column was determined with blue dextran. 

The selectivity of a column is primarily a function of the packing material, although the chromatographer has some control using the mobile phase or temperature. The value for a can range from unity (1), when the retention times of the two components are identical (t2 = fi), to infinity if the first component of interest is eluted in the void volume. If a is approaching 1,... 

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