Void volume determination

Chromatorapf c methods For gel filtration of polysaccharide fraction PI, a Sephacryl S-300 chromatographyc column (1,1 X 46,7 cm) was calibrated with standard dextrans (molecular mass range 266, 72, 40, and 17 KDa Sigma Chemicals), and the void volume determined with blue dextran. Polysaccharide sample (0.5 mL 2 mg/mL) was applied and eluted with 50 mM NaOH, fractions 1 mL being collected and carbohydrate absorbance (phenol-H2S04) being monitored. 

Experimental assessment of the column void volume proved to be critical since the solute retention volume approaches the void volume as pressure is increased. Following the recommendations of Kobayashi (24), we used an unretained solute, methane, for this measurement. Values for the void volume determined over an extended pressure range were 1.8 and 0.5 ml. for the crosslinked resin and alumina columns, respectively. These figures were in excellent agreement with void volume approximations of 1.4 and 0.45 ml. based upon the geometric volume of the column assuming a porosity of 0.6 for the packed beds. 

Void volume is a critical parameter in HPLC, since most theoretical relationships in chromatographic theory deal with the retention factor, and the accuracy of the void volume determination plays an important role in all calculations. 

In most analytical applications of HPLC, all these discrepancies are quietly and conveniently forgotten, and selection of some so-called nonretained component as a void volume marker is a common way for void volume measurement. In the majority of recent analytical publications, either thiourea or uracil were used as the void volume markers. As a disclaimer, we have to say here that for the purposes of analytical method development, qualitative or quantitative separation of complex mixtures which involves the use of a nonretained component as a void volume marker is acceptable insofar as there are no physicochemical generalization, thermodynamic development, or futher theoretical development performed upon the basis of these pseudo void volume determinations. 

As a simple illustration of the inconsistency of the use of any nonretained components for the void volume determination, we remind the reader that in the basic assumptions and in the derivation of expressions (2-43) and (2-46), void volume of the column was considered to be constant in any eluent type and composition. Figure 2-10 illustrate the dependence of the thiourea and uracil retention in acetonitrile/water and in methanoFwater as a function of organic content in the eluent. The horizontal line in Figure 2-10 shows the... 

In everyday method development practice, it is important to ensure the separation of target compounds, matrix components, and other impurities. The elution of the analyte at the void volume means that it did not interact with the stationary phase and thus could not be separated from other components that do not interact with the surface either. To ensure the analyte interaction with the stationary phase, it is usually recommended to choose chromatographic conditions when any component of interest elutes with at least 1.5 void volume values or even greater. The error of the void volume determination for these purposes could be 20% or even greater (insofar as these void volume values are not used for any calculations but just to estimate where the least retained analye elutes). The use of uracil, thiourea, or allantoin as analytical void volume markers is most common in practical analytical work. 

Ion interaction in the stationary phase [15] and intramolecular hydrophobic interactions [16] have been mentioned as other possible reasons for the occurrence of system peaks. Knox and Kaliszan [17] have discussed the origin of system peaks in their classical investigation of the methods of void volume determination. 

Key = Void volume determined from water content, CEC = Cation exchange capacity. 

Gel Filtration. The lyophilized protein was redissolved in 50 mM phosphate buffer, pH 7.4 0.15 m NaCl 0.013 % sodium azide and loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fi actions were collected. Fractions with pectin lyase activity were combined, dialyzed against distilled water and used in the next step. To estimate the molecular mass of PNL, the column was calibrated with standard proteins (Sigma MW-GF-70 Albumin, 66,000 Da Carbonic Anhidrase, 29,00 Cytochrome, 12,400 and Aprotinin, 6,500). The proteins were eluted in the conditions described above and their volumes (F ) were calculated fi om the peak maximum of the absorbance at 280 nm. The partition coefficient was obtained fi om the relationship where F, represents the bed volmne of column and F the void volume (which was calculated using blue dextran. Sigma). The molecular mass was determined using a standard curve of vs the logarithm of the molecular masses of the standards [28, 29]... 

This method determines the pore volume corresponding to pore radii below 75,000 A. By varying the pressure on the system, it is possible to force the mercury into some of the pores and determine the void volumes corresponding to different pore radii. We will pursue this point in the next section. 

A catalyst for cracking cumene is available commercially in the form of 0.35 cm diameter pellets. These pellets have a specific surface area of 420 m2/g and a void volume of 0.42 cm3/g. If the apparent first-order rate constant for this reaction is 1.49 cm3/sec-g catalyst at 412 °C, determine the effectiveness factor of the catalyst. 

It is a great deal of work to actually determine a true equilibrium constant and most chemical separation methods speak in terms of values which are proportional to the actual equilibrium constant. At constant flow, the time that a given type of molecule is retained is related to the time for the void volume to pass after the sample is placed in a column or on a plate with the addition of the time for the net retention volume. If the flow remains constant, the temperature of the separation remains constant and no stationary phase is gained or lost, one can attempt qualitative identification using retention times. It is more reasonable to calculate the ratio of net retention volume to the void volume and call the result partition factor or capacity factor, k. ... 

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