How do SEC columns work?

How do SEC columns work?

SEC works by trapping smaller molecules in the pores of the adsorbent (“stationary phase”). This process is usually performed within a column, which typically consists of a hollow tube tightly packed with micron-scale polymer beads containing pores of different sizes.

How do you choose a SEC column?

How to Choose the Right SEC Column for Your Analysis

1. Pore size and column dimension is based on the complexity of sample to be separated.
2. LC System dispersion used for the analysis, as well as sample throughput.
3. Based on these criteria, the most appropriate UPLC, UHPLC, or HPLC offering can be selected.

How do you choose a column for size exclusion chromatography?

Select a column with a bed height providing the required resolution. A bed height between 30 and 100 cm is recommended for preparative separation. Select a column size appropriate for the volume of sample that needs to be processed. Select the highest flow rate that maintains resolution and minimizes separation time.

What is Protein SEC?

Size-exclusion chromatography (SEC), also known as gel filtration, separates proteins based on their sizes (hydrodynamic radii versus absolute molecular weight) in solution with larger sized species eluting before smaller proteins.

Which solvent is used for SEC?

There are two types of SEC: One uses organic solvent and the other uses aqueous solvent as a mobile phase. The one uses aqueous solvent is called Gel Filtration Chromatography (GFC) or aqueous SEC.

What does RP HPLC do?

RP-HPLC is a commonly used method for the analysis and purification of peptides, proteins, and glycoproteins. Monosaccharide composition and content can be determined by using the RP-HPLC separation of p-aminobenzoic ethyl ester derivatives of neutral and amino sugars released from glycoproteins.

What does equilibrate the column mean?

Column Equilibration A buffer that is compatible with the protein of interest and the resin of choice is passed over the column. A common practice is to equilibrate the column with 5–10 column volumes (CVs) of equilibration buffer.

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.

How is column chromatography done?

Column chromatography is a versatile purification method used to separate compounds in a solution. A solution mixture is carried by a solvent through a column containing an adsorbent solid, called the stationary phase. Compounds that interact weakly with the stationary phase are faster to exit the column, or elute.

What is SEC analysis?

Size exclusion chromatography (SEC or SEC-HPLC) for measurement of protein absolute molecular weight, structure, size and conformation. Size Exclusion Chromatography (SEC or SEC-HPLC) is an analytical technique that separates dissolved macromolecules by size based on their elution from columns filled with a porous gel.

How does SEC chromatography work?

Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a gel. The gel consists of spherical beads containing pores of a specific size distribution. Separation occurs when molecules of different sizes are included or excluded from the pores within the matrix.

On what basis are proteins separated by SEC?

Size exclusion chromatography
Size exclusion chromatography (SEC), also called gel filtration, gel permeation, molecular sieve, and gel exclusion chromatography, is a separation technique used to separate molecules on the basis of size and shape (hydrodynamic radius).

Which is the best size for SEC columns?

Particle size of the SEC packing media as well as column length are important parameters that determine separation efficiency. BioSuite 4 μm particle size, UHR Columns provide maximum separation efficiency, followed by BioSuite HR Columns and BioSuite Standard SEC Columns.

How does SEC work in a chromatography column?

SEC is an entropically-controlled separation process in which the analytes are “filtered” rather than retained on the column through chemical interactions between the stationary phase and specific, functional groups on the analyte.

Why do we use Beh SEC column technology?

As the molecular weight of the proteins of interest increases, SEC columns containing comparatively larger pore size particles are selected. Why choose BEH SEC column technology?

Which is the best SEC column in BIOSuite?

BioSuite 4 μm particle size, UHR Columns provide maximum separation efficiency, followed by BioSuite HR Columns and BioSuite Standard SEC Columns. To maximize column life of analytical (i.e., 4.6 mm or 7.8 mm I.D.) or preparative (i.e., 21.5 mm I.D.) SEC columns, use of BioSuite Guard Columns is recommended.