Volume elution

Elution volume, exclusion chromatography Flow rate, column Gas/liquid volume ratio Inner column volume Interstitial (outer) volume Kovats retention indices Matrix volume Net retention volume Obstruction factor Packing uniformity factor Particle diameter Partition coefficient Partition ratio Peak asymmetry factor Peak resolution Plate height Plate number Porosity, column Pressure, column inlet Presure, column outlet Pressure drop... 

Dirac delta function S Elution volume, exclusion Vo... 

Fig. 8. Representation of measurement of elution volume, as a function of sample volume (a) <2% of bed volume, (b) >2% and (c) >2% and giving a plateau region which has the same concentration as the iajected sample A represents the inflection poiat. See text.

The elution volume, F/, and therefore the partition coefficient, is a function of the size of solute molecule, ie, hydrodynamic radius, and the porosity characteristics of the size-exclusion media. A protein of higher molecular weight is not necessarily larger than one of lower molecular weight. The hydrodynamic radii can be similar, as shown in Table 4 for ovalbumin and a-lactalbumin. The molecular weights of these proteins differ by 317% their radii differ by only 121% (53). 

Liquids have relatively low compressibility compared with gases and, thus, the mobile phase velocity is sensibly constant throughout the column. As a consequence, elution volumes measured at the column exit can be used to obtain retention volume data and, unless extreme accuracy is required for special applications, there is no need for the retention volume to be corrected for pressure effects. 

Calibrate the system. Use narrowly dispersed molecular weight standards of the polymer of interest to construct a calibration curve of log molecular weight versus elution volume (Eig. 3.2). If a more sophisticated software system is available, a broad molecular weight standard may be used to calibrate the system. 

The elution volume of a molecule in HPSEC is determined by its hydrodynamic size and the pore size of the column packing. In setting up an HPSEC experiment, the chromatographer must match the pore size of the column to the molecular size range of the sample. 

Errors in the molecular weight data from HPSEC are usually due to improperly prepared samples, column dispersity, or flow rate variations. The sample to be analyzed should be completely dissolved in the mobile phase and filtered prior to injection onto the column. A plugged column inlet frit will invalidate results. In addition, do not load the column with excess sample. Column overloading affects the accuracy of data by broadening peaks, reducing resolution, and increasing elution volume. For best results, the concentration of the injected sample should be as low as possible while still providing adequate... 

Tailing peaks or longer than expected elution volumes are sometimes caused by low solubility of the protein in the mobile phase. Using a trial-and-error process, select the proper pFf and ionic strength to address this problem. Detergents such as sodium dodecyl sulfate (SDS) are sometimes helpful but, because they change the conformation of many proteins and are difficult to remove from the column should be used only if other methods fail. 

Sample/remark Water 7.0 Elution volumes in buffered solutions (pH) 5.5 6.8 8.5 ... 

TABLE 4.9 Comparison of Peptide Elution Volumes on TSK-GEL G2500PWxl and TSK-GEL G2000SWxl Columns... 

FIGURE 4.29 Relation between molecular weight of lipoproteins and elution volume for combination GFC columns. Column 7.5 mm i.d. X 60 cm. Sample Chylomicron, VLDL, LDL, HDLj, HDL3, albumin, and ovalbumin. Elution 0.1 hA Tris—HCI buffer (pH 7.4). Flow rate 1.0 ml/min. 

FIGURE 4.43 Calibration curves for globular proteins on toyopearl resins. Column 22 mm X 30 cm. Sample Protein standards. Elution 0.06 A1 phosphate buffer, pH 7, in 0.06 A1 KCI. Legend elution volume V column volume. 

The elution volumes of Dextran Blue or DNA are equivalent to the void volume (Vo) of the column. Usually, a small difference appears between the two values due to differences in molecular shape and slight nonspecific interaction with the matrix. 

The elution volume of sodium nitrate [ Ve (NaN03)] allows one to calculate the volume of solid matrix (V ,) in the column according to... 

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