High Pressure Size Exclusion chromatography (HPSEC) is increasingly used to evaluate molecular sizes of humic substances from different sources. HPSEC is confirmed to represent a highly precise method to evaluate the relative molecular-size distribution of dissolved humic substances.
What is high performance size exclusion chromatography?
High performance size-exclusion chromatography (HP-SEC) is a well-established, robust and highly versatile technique for the characterization and quantification of protein monomers, fragments and aggregates. In HP-SEC, the sample is injected onto a column that is constantly flushed with mobile phase.
Why is size exclusion chromatography important?
The purpose is to separate small molecules, such as salts, from large biomolecules such as proteins. Samples can be prepared for storage or for other chromatography techniques and assays. Large sample volumes—up to 30% of the total column volume—can be applied at high flow rates using broad, short columns.
Why is gel filtration chromatography important?
One of the principal advantages of gel-filtration chromatography is that separation can be performed under conditions specifically designed to maintain the stability and activity of the molecule of interest without compromising resolution.
What is the principle of GPC?
GPC is based on separation by molecular size rather than chemical properties. It employs the principle of size-exclusion chromatography (often referred to as SEC) to separate samples of polydisperse polymers into fractions of narrower-molecular-weight distribution.
What buffers is used in size exclusion chromatography?
Buffers with pH between 6.0 and 8.0 are normally used in SEC applications since many proteins are stable in that pH range. A running buffer of 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.0 works well for many SEC separations.
What is the principle of gel filtration?
Principle. The gel filtration chromatography is based on the molecular size and the hydrodynamic volume of the components. The molecules are separated by the differential exclusion or inclusion of solutes as they pass through the stationary phase containing heteroporous cross-linked polymeric gel or beads.
What is the column used in gel filtration chromatography?
Gel filtration is performed using porous beads as the chromatographic support. A column constructed from such beads will have two measurable liquid volumes, the external volume, consisting of the liquid between the beads, and the internal volume, consisting of the liquid within the pores of the beads.
What does GPC measure?
Gel Permeation Chromatography (GPC) for measurement of polymer molecular weight, size and structure. Gel Permeation Chromatography (GPC) is an analytical technique that separates dissolved macromolecules by size based on their elution from columns filled with a porous gel.
What is the difference between SEC and GPC?
It is exactly the same. Just two different ways to name it. It is the same thing. Gel permeation chromatography (GPC) is a type of size exclusion chromatography (SEC).
What is blue dextran?
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.
Is gel filtration the same as size exclusion?
Gel filtration chromatography, also known as size exclusion chromatography, is used to separate molecules of different sizes. In addition to separating different proteins of varying size, one may resolve oligomeric forms of a particular protein.
What column is used for hpsec analysis?
For HPSEC analysis, a ThermoElectron BioBasic SEC60 column, with pore size 60 Å, pore volume 0.7 ccg-1 and particle size 5 µm, is used. The column consists of 5 µm silica particles, coated with a hydrophilic polymer to ensure separation solely based on solute size.
What is high performance size-exclusion chromatography (hp-SEC)?
High performance size-exclusion chromatography (HP-SEC) is a well-established, robust and highly versatile technique for the characterization and quantification of protein monomers, fragments and aggregates. In HP-SEC, the sample is injected onto a column that is constantly flushed with mobile phase.
What is hp-SEC and why do you need it?
HP-SEC is commonly employed in all stages of research and development of biopharmaceuticals. It is highly valuable during formulation development, stability studies and forced degradation studies, but is most frequently used for batch release testing. Coriolis offers HP-SEC also in full GMP compliance.
What is hp-SEC column made of?
In HP-SEC, the sample is injected onto a column that is constantly flushed with mobile phase. The chromatography column itself contains fine and porous beads, which are composed of dextran, agarose, silica or polyacrylamide (the stationary phase).
