Sephadex column, elution volume

Water-soluble polymeric dyes have been prepared from water-insoluble chromophores, viz., anthraquinone derivatives. Unreacted chromophore and its simple derivatives, which are all water-insoluble, remain in solution due to solubilization by the polymeric dye. A method has been developed to separate and quantitate the polymeric dye and these hydrophobic impurities using Sephadex column packing. The solvent developed has the property of debinding the impiirities from the polymer, and further allows a separation of the imp irities into discrete species. This latter separation is based on the functional groups on the impurity molecules, having a different interaction with the Sephadex surface in the presence of this solvent. The polymer elutes at the void volume... 

Briefly, liposomes (10mM) were incubated for 30minutes at 37°C for egg phosphatidylcholine (EPC) and at 60°C for HSPC-based liposomes with 50 X 10 dpm of methylamine (1 x 10 dpm/mole). At the end of incubation an aliquot of this mixture was passed down a Sephadex G-50 minispin column equilibrated in 10 mM histidine-sucrose buffer 10%, pH 6.7 buffer. Liposomes were eluted at the column void volume and separated from the unencapsulated methylamine. The concentration of liposomes in the original liposomal dispersion and in the void volume fraction was determined from the organic phosphorus (phospholipid) concentration (see section Lipid Quantification and Chemical Stability above) (10,49,53). 

Void volume is measured by passing a large, inert molecule through the column." Its elution volume is defined as F0. Blue Dextran 2000, a blue dye of molecular mass 2 X 106, is commonly used for this purpose. The volume Vm can be calculated from the measured column bed volume per gram of dry gel. For example, 1 g of dry Sephadex G-100 produces 15 to 20 mL of bed volume when swollen with aqueous solution. The solid phase occupies only 1 mL of the bed volume, so Vm is 14 to 19 mL, or 93-95% of the total column volume. Different solid phases produce widely varying column bed volumes when swollen with solvent. 

Figure 3-9 Elution volume of various proteins on a column of Sephadex G-200 as a function of molecular mass. The right-hand vertical axis shows the ratio of the elution volumes to that of blue dextran, a high-molecular-mass polysaccharide that is excluded from the internal volume. After Andrews.193...

Isoenzymes. Multiple forms of citrus PE were reported by Evans and McHale (AO) and Versteeg et al. (Al). PE was purified from West Indian limes and Navel oranges by fractionation of the whole fruit extracts with (NH. SO (AO-65%), adsorption and elution from Sephadex G-75 columns (AO). The PE active fractions were combined and concentrated before separation into two active PEs on the basis of their elution volume from a DEAE Sephadex A-50 column. Orange PEI (OPEI) and lime PEI (LPEI) had the same elution volume also OPEII and LPEII had the same elution volume. A higher concentration of NaCl was required at all pH values for optimum activity of OPEI and LPEI than of OPEII and LPEII. When the component parts of oranges were separately analyzed chromatographi-cally with DEAE-Sephadex A-50, OPEI was detected only in the peel, whereas OPEII was identified in juice sacs and section walls (AO). 

Fig. 4-2 Gel filtration of proteins. Elution volumes Vcl are plotted for a series of proteins of different Mr applied to a column of Sephadex G-200. The two data points denoted by were not used in constructing the calibration curve.

Gel filtration of the hemicellulose was done on a G-50 Sephadex column with water as eluent by the National Food Research Institute of the CSIR. Neutralized samples were applied to the column and fractions (6 mL) collected, of which 1-mL aliquots were assayed by the phenol-sulfuric acid test ( ). Approximate calibration (D.P. versus elution volume on the G-50 column) was done by total carbohydrate and reducing sugar end group analysis (0) using enzymatically debranched starch as reference material. 

Figure 2-6 Calibration of a Sephadex G-200 gel filtration column. V,/V, represents the elution volume of a given protein relative to the elution volume of a very large molecule that is completely excluded from the gel. The standard proteins are more or less spherical and have average amino acid contents.

A clean separation of the 4 main nucleosides (either ribo- or deoxyribo-) on cross-linked Dextran (Sephadex G-10) has been described by Braun (1967b). He used a 1.5x90 cm column eluted with 0.01 M citric acid Na2HP04, pH 3.5 at 25 ml/hr. The sample was loaded in 0.5 ml water and contained 0.1 mg of each nucleoside. The elution took 10 hr and each nucleoside was eluted in a volume of 10-20 ml in the order, C, U, A, G or C, T, A, G for the... 

Fig. 39. Comparative elution volumes of proflavine and blue dextran under different conditions of solvent and temperature with Sephadex LH-20 column (2 x 2.5 cm). Equilibration of the column is performed with the elution medium 1 ml of a 0.3...

The serum stability was studied by incubation of I-DOTATATE in human serum overnight at ambient temperature. Determination of serum protein binding was carried out by gel permeation chromatography using a Sephadex G-25 column (2.5 cm x 30 cm) and elution using 0.05M phosphate (elution volume 1 mL fractions). The l-DOTATATE was incubated in serum at ambient temperature for 18 h and then treated with 10% trichloroacetic acid for precipitation of the protein bound fraction. The supernatant was tested by HPLC to estimate its purity. 

Molecular sieves are useftil for the desalting of proteins that have been partially fractionated by salting out with a high concentration of some salt. A gel with a low exclusion hmit, such as Sephadex 25, will allow the proteins to pass right through the column while the salts are retained. The protein dilution is limited to the elution volume of the column (the volume external of the swelled gel to fill the column). 

The elution volume from a Sephadex column is a monotonically decreasing function of molecular weight. Therefore, by comparing the elution behavior of an unknown protein with that in a series of standards of known molecular weight, an estimation of molecular weight can be interpolated. 

A series of standard proteins and an unknown enzyme were studied by gel filtration on a Sephadex G200 (the 200 refers to the maximum pore size size in kDa) column. The elution volume Vei for oach protein is given below, (a) Plot the data in the form of log versus elution volume, (b) Erom the line of best fit through the points for the standards, determine the of the unknown enzyme, (c) Explain why ferritin and ovomucoid behave anomalously. The Vj,i versus data were as follows ... 

Fig. 4-24 Filtration of proteins from a column of Sephadex G200. The elution volume Vei is plotted for a series of different proteins of known that had been applied in a mixture to the column.

The effect of Blue Dextran on protection from insolubilization of NaCl-released 3-D-fructofuranosidase from sugar beet cell-wall was studied and discussed.The enzyme was insoluble in water or salt solutions of low concentrations but was solubilized by the addition of Blue Dextran. Upon chromatography of the enzyme on a Sepharose 6B column in the presence of Blue Dextran, the enzyme and Blue Dextran were eluted with the same elution volume. However, they were eluted separately by using 0.5 M-NaCl as an eluting solvent. Solubility of enzyme increased with concentration of Blue Dextran added. It was found that the enzyme was absorbed to Sephadex G-lOO, but not to dextran T40 or T500. Some additional experiments revealed that the dependence of pH on solubility of the released enzyme disappeared in the presence of Blue Dextran. 

In an article exposing their results [12], they were the first to use the Martin and Synge theoretical derivation to estabhsh the equation relating the solute retention to its micellar partition coefficient. Although their Sephadex G-25 column presented a strong adsorption for some solutes, they were able to obtain straight lines plotting the Vi/(Ve-Vo) parameter versus the SDS surfactant concentration, the subscripts i, e and o refer to the internal (pore) volume of the GPC gel, the solute elution volume and the external (exclusion) volume, respectively. The slope and intercept of the lines allowed them to determine accurately the micellar partition coefficient of the seven compounds studied [12]. They were not interested in the separation ability of the technique and did not go further in this direction. 

When the SEC of lignins is performed on an open column packed with soft gels, such as Sephadex G, the elution volume of the same solute size varies slightly between runs due to small fluctuations in the flow rate and ambient temperature that may disturb the void volume and total permeation volume of the gel bed. Moreover, a new column has to be packed every three months for a Sephadex G gel/aqueous 0.1 M NaOH system because reproducible column performance can be maintained only for a finite period of time as a result of slow decomposition of the packing material. Thus, the variation in the elution volume between columns can be considerably large because of variation in the height of the gel bed from one column to another. To overcome uncertainty arising from these potential sources, the elution volume is often converted to a dimensionless variable prior to y-axis... 

The values of Vj and Vo are usually determined by the respective standard markers. For example, in the Sephadex G-lOO/aqueous 0.1 M NaOH system, blue dextran (BD) and p-nitrophenol (pNP) are often used as standard markers for Vo and Vj, respectively. In the standardized elution profiles depicted in Fig. 2, the elution volume has been converted to a slightly different dimensionless parameter, termed relative retention volume (Vr), which is operationally defined as Vr = Vj x scaling factor, where Vr = (V — Vo)/Vo. The Vb is taken as the starting, rather than the apex position, of the BD peak. " The scaling factor normalizes Vr, which may vary slightly from ran to ran due to experimental background variations, by setting the Vr value of pNP to 2 (since the Vr value of pNP eluted from a 2.5 x 100 cm column packed with Sephadex G-lOO with aqueous 0.1 M NaOH is close to 2). Thus, an equivalent expression is Vr = 2 x (V — Vq)/ (K,np - Vo), where VpNp is the elution volume of... 

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