Exclusion chromatography void volume

In molecular exclusion chromatography, the volume of mobile phase (the solvent) in the column outside the stationary phase is called the void volume, Vq. Large molecules that are excluded from the stationary phase are eluted in the void volume. Void volume is measured by passing through the column a molecule that is too large to enter the pores. The dye Blue Dextran (2 X 10 Da) is commonly used. 

In 1971, Hiatt et al. found that polyethylene oxide (PEO) of molecular weight about 100000 prevented the adsorption of rabies virus to porous glass with an average pore diameter of 1250 A. The support was modified by passage of one void volume of 0.4% solution of the polymer in water, followed by 5 or more volumes of distilled water or buffered salt solution. The virus was effectively purified from the admixtures of brain tissue fluid by means of size-exclusion chromatography on the modified glass column [28]. 

Size exclusion chromatography has been used to analyse the size distribution of liposomes. For example, SUV can be separated from MLV, which elute in the void volume, by using a Sepharose 4B gel. 

In exclusion chromatography, the total volume of mobile phase in the column is the sum of the volume external to the stationary phase particles (the void volume, V0) and the volume within the pores of the particles (the interstitial volume, Vj). Large molecules that are excluded from the pores must have a retention volume VQ, small molecules that can completely permeate the porous network will have a retention volume of (Vo + Fj). Molecules of intermediate size that can enter some, but not all of the pore space will have a retention volume between VQ and (V0 + Fj). Provided that exclusion is the only separation mechanism (ie no adsorption, partition or ion-exchange), the entire sample must elute between these two volume limits. 

Particulate sorbents are available almost exclusively in the shape of micrometersized beads. These beads are packed in columns and represent currently the most common stationary phases for high-performance liquid chromatography (HPLC). Despite their immense popularity, slow diffusional mass transfer of macromolecular solutes into the stagnant pool of the mobile phase present in the pores of the separation medium and the large void volume between the packed particles are considered to be major problems in the HPLC of macromolecules, frequently impairing their rapid and efficient separation [1]. 

We foimd that the ratiometric method is superior because it is not dependent on pyranine concentration and therefore free of error in pipeting (18,22,54). Calibration curves were constructed by preparing liposomes in which the hydration of the lipids to form MLV was done using solutions of high concentration at the desired pH in the range of 3.0 to 10.0. Gel-exclusion chromatography on a Sephadex column, as mentioned above, yielded a series of liposome preparations with a fixed external pH (pH 7.5), but different internal pH values determined by the buffer used for lipid hydration. Neither KI nor DPX, which quench the fluorescence of aqueous solutions of pyranine, has much effect on the fluorescence intensity of pyranine in the void volume after gel-exclusion chromatography, which indicates the complete removal of the pyranine from the extraliposomal medium. 

Y = ratio between [ C]-methylamine counts (dpm) and phospholipid concentration (mM) in the void volume fraction after the separation by gel-exclusion chromatography. Percentage of encapsulation (%) = Y/Xx 100. 

Ferritin (molecular mass 450 000), transferrin (molecular mass 80 000), and ferric citrate were separated by molecular exclusion chromatography on Bio-Gel P-300. The column had a length of 37 cm and a 1.5-cm diameter. Eluate fractions of 0.65 mL were collected. The maximum of each peak came at the following fractions ferritin, 22 transferrin, 32 and ferric citrate, 84. (That is, the ferritin peak came at an elution volume of 22 X 0.65 = 14.3 mL.) Assuming that ferritin is eluted at the void volume and that ferric citrate is eluted at Vm, find Kay for transferrin. 

The separation range in size-exclusion chromatography for a particular column is relatively narrow, and it lies between the total volume of the liquid phase in the column (void volume) and the exclusion volume, Ve. The difference between these two volumes is the total pore volume of the packing material in the column. Indeed, if some molecules of studied polymers are small enough to penetrate inside all pores of the packing material, they will elute with the column void volume. On the other hand, polymers with significant molecular size that cannot penetrate inside the particles will all travel together around the particles and elute early with exclusion volume. 

Separation in size-exclusion chromatography requires careful matching of the pore size of the stationary phase material with the size of the molecules to be separated. Small molecules in a sample will be able to penetrate all the pores of the stationary phase and will elute with an elution volume, Fe, which is equal to the void volume of the column, Fm- Very large molecules will be excluded from all the pores of the stationary phase and will elute with an elution volume, which is equal to the interstitial volume of the Uquid between the particles, Fj. Molecules of intermediate size will be able to penetrate some but not all of the pores and will elute with an elution volume that is between the interstitial volume and the void volume of the column. The void volume is the total volume of the liquid in the column and is related to the interstitial volume and the pore volume, Fp by equation (3.38) ... 

The introduction of ion-exclusion chromatography is attributed to Wheaton and Bauman [1], It serves, above all, for the separation of weak inorganic and organic acids. In addition, ion-exclusion chromatography can be utilized for the separation of alcohols, aldehydes, amino acids, and carbohydrates. Due to Donnan exclusion, fully dissociated acids are not retained at the stationary phase, eluting therefore within the void volume as a single peak. Undissociated compounds, however, can diffuse into the pores of the resin, since they are not subject to Donnan exclusion. In this case, separations are based on non-ionic interactions between the solute and the stationary phase. 

The elution problem does not occur in size-exclusion chromatography, which is one of its major advantages. Instead, elution of all components is guaranteed, the final ones coinciding with the void volume at the very latest. The only exception to this is in the case of adsorption effects, peaks being eluted later than Vq, but this can be prevented by a careful choice of stationary and mobile phases. 

To understand how steric exclusion differs from the other forms of chromatography, refer to Equation 21.4. In this context, V a and Va are referred to as the void volume and the total pore volume, respectively. The distribution coefficient depends on the molecular weight of the sample and on the pore size of the packing. The equilibrium established in exclusion chromatography is described by Equation 21.1 ATx is defined by Equation 21.2. In a true permeation process, assuming all pores to be accessible to a small solute molecule, and = 1- If none of the pores... 

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