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Why is blue dextran used in gel filtration chromatography?

Why is blue dextran used in gel filtration chromatography?

Blue dextran: It is too large and can’t get into the beads and therefore excluded from the gel (Kd=0), and pass the column in the void volume (space between gel beads). Blue dextran is often used as a marker to measured void volume.

How does gel filtration chromatography work?

Gel filtration (GF) chromatography separates proteins solely on the basis of molecular size. Separation is achieved using a porous matrix to which the molecules, for steric reasons, have different degrees of access–i.e., smaller molecules have greater access and larger molecules are excluded from the matrix.

Which compounds elute first in gel chromatography?

During chromatography, small molecules may diffuse out of the mobile phase into the pores so their progress is retarded relative to that of larger molecules. The smaller the molecule, the further it will be able to penetrate into the pores and the more it will be retained, so the largest molecules elute first.

Why large molecules elute first in gel filtration?

Gel filtration chromatography (also called size exclusion chromatography) employs porous beads with a defined pore size distribution as the stationary phase. Small molecules can enter the entire intraparticular pore space and hence elute last, whereas large molecules are excluded from all pores and hence elute first.

What is dextran blue?

Blue Dextran is a high-molecular-weight glucose polymer (original mol wt 2 x 10(6) g/mol) containing covalently bonded Reactive Blue 2 dye (approximately mmol/g dextran). This blue dye is known for its high binding affinity to a wide variety of proteins, with a particularly high affinity for serum albumin.

Why did the blue dextran and the DNP glycine separate in the column?

Blue dextran and DNP-glycine are separated on the basis of molecular weight by gel permeation (Biogel P100) chromatography. Adherent macromolecules are subsequently eluted from the columns. The presence of each type of protein is verified on the basis of color or, in the case of albumin, absorbance at 280 nm.

Where is gel filtration chromatography used?

Gel filtration chromatography can be used to separate compounds such as small molecules, proteins, protein complexes, polysaccharides, and nucleic acids when in aqueous solution. When an organic solvent is used as the mobile phase, the process is instead referred to as gel permeation chromatography.

What is the significance of using blue dextran in molecular exclusion chromatography when is it used and why?

Blue dextran is also used for checking the column packing. A symmetrical peak of elution indicates homogeneity of packing. The inner volume (Vi) of the column can be determined by subtracting the void volume from the elution volume of small molecules such as glucose or tyrosine having K d — 1.0.

What is the function of dextran?

Dextran is a medication used in managing and treating various clinical conditions, including during hemorrhage, shock, surgical procedures, radiological imaging, antithrombotic administration, and ophthalmic relief of xerophthalmia.

What is the size of blue dextran?

∼2,000 kDa
The Vo of a given column is based on the volume of effluent required for the elution of a large molecule such as blue dextran (molecular mass of ∼2,000 kDa, Catalog Number D4772).