size exclusion chromatography

Separate biological molecules and polymers according to size

Size exclusion chromatography (SEC) is a major mode of HPLC that employs porous particles in the column to separate molecules by virtue of their size in solution. SEC is generally used to separate biological molecules, to determine molecular weight distributions of proteins and peptides, as well as to separate a long list of water soluble polymers used in a wide range of industries. 


Size exclusion columns for HPLC

  MAbPac SEC-1 HPLC Columns BioBasic SEC HPLC Columns Acclaim SEC HPLC Columns
Applications Optimized for monoclonal antibodies aggregates, monomers, fragment separation Various biologics including peptides and proteins, some water soluble polymers Water soluble polymers and oligomers
Base material Silica based Silica based Polymer based
pH range pH = 2.5 to 7.5 pH = 2 to 8 pH = 2 to 12
Pore size 300Å 60Å, 120Å, 300Å, 1000Å 300Å, 1000Å
Particle size 5µm 5µm 5µm and 7µm

Choose from three brands of SEC columns depending on your application:

MAbPac SEC HPLC columns for monoclonal antibody, aggregate, and fractions separation

MAbPac SEC-1 columns are specifically designed for the optimal, high-resolution separation and characterization of monoclonal antibodies (mAbs), monomers, aggregates, and fragments.

  • Analysis of monoclonal antibodies (mAbs) and their aggregates
  • Analysis of mAb Fab and Fc fragments, even using high and low salt concentrations
  • Hydrophilic bonded layer for minimal non-desired interactions between the biomolecules and the stationary phase
  • Stable surface bonding leads to low column bleed and compatibility with MS, ELSD, and Corona Charged Aerosol Detection (CAD)
  • Separation range for globular proteins 10,000−1,000,000; exclusion limit for globular proteins >1,000,000
MAbPac SEC-1

MAbPac SEC 1 HPLC Columns

Particle size Description ID (mm) Length (mm) Cat. No.  
5µm Guard Column 4 50 074697 Order
5µm HPLC Column 2.1 150 088790  Order
5µm HPLC Column 2.1 300 088789 Order
5µm HPLC Column 4 150 075592 Order
5µm HPLC Column 4 300 074696 Order
5µm HPLC Column 7.8 300 088460 Order

BioBasic SEC HPLC columns for protein and peptide separation

  • Superior separation of various biologics including peptides and proteins.
  • Separates analytes over a wide molecular weight range
  • Long column life and high column efficiencies
  • Simple mechanism of interaction based on molecular size and shape
  • Ideal for sample clean-up; often used as the first step in isolation of a protein from a crude sample.
  • Straightforward method development, simple mobile phases
  • When used with standards for calibration, it is possible to determine the molecular mass of proteins.
  • These columns can also be used for water soluble polymers
BioBasic SEC

Which BioBasic size exclusion column to choose

This chart provides molecular weight ranges (KDaltons) for BioBasic SEC columns

Phase Pore Size Proteins Pullulans Polyethylene Oxides/Glycols
BioBasic SEC 60 60Å 0.1–6 0.3–6 0.1–4
BioBasic SEC 120 120Å 0.3–12 0.3–100 0.4–10
BioBasic SEC 300 300Å 1–500 20–>500 2–100
BioBasic SEC 1000 1000Å 20–4000 20–>1000 Not Recommended
Pore Size (Å) Description ID (mm) Length (mm) Cat. No.  
BioBasic SEC 60
60 Guard Column 7.8 30 73305-037821 Order
60 HPLC Column 7.8 150 73305-157846 Order
60 HPLC Column 7.8 300 73305-307846 Order
BioBasic SEC 120
120 Guard Column 7.8 30 73405-037821 Order
120 HPLC Column 7.8 150 73405-157846 Order
120 HPLC Column 7.8 300 73405-307846 Order
BioBasic SEC 300
300 Guard Column 7.8 30 73505-037821 Order
300 HPLC Column 7.8 150 73505-157846 Order
300 HPLC Column 7.8 300 73505-307846 Order
BioBasic SEC 1000
1000 Guard Column 7.8 30 73605-037821 Order
1000 HPLC Column 7.8 150 73605-157846 Order
1000 HPLC Column 7.8 300 73605-307846 Order

Acclaim SEC HPLC columns for water soluble polymers

  • Separation of water soluble polymers and oligomers
  • Water soluble polymers are a highly varied family of products including glycols, polyvinyl alcohols, polyvinyl pyrrolidones, dextrans, polyacrylic acids, and more.
  • The Acclaim SEC-1000 has a nominal pore size of 1000 Å for separating polymers and oligomers in the MW range of 1,000
    to 1,000,000 Dalton
  • The Acclaim SEC-300 has a nominal pore size of 300 Å for separating in the MW range of 100 to 50,000 Dalton  

pH range 2-12
Pore size 300, 1000
Particle size 5µm, 7µm
Temperature <60°C 

Which Acclaim SEC column to choose

  Acclaim SEC-300 Acclaim SEC-1000
Substrate Hydrophilic polymethacrylate resin Hydrophilic polymethacrylate resin
Particle shape Spherical Spherical
Particle size 5μm 7μm
Pore size 300Å 1000Å (multi-pore)
Separation range for PEO* 100–50,000 Daltons 1,000–1,000,000 Daltons
Exclusion limit for PEO* 50,000–150,00 Daltons 3,000,000–7,500,000 Daltons

*PEO = polyethylene oxides

Pore Size (Å) Particle size Description ID (mm) Length (mm) Sample Loading (µL) Cat. No.  
Acclaim SEC-300 LC Columns
300 5µm Guard Column 4.6 33 - 082740 Order
300 5µm HPLC Column 7.8 150 <150 079726 Order
300 5µm HPLC Column 7.8 300 <300 079725 Order
300 5µm HPLC Column 4.6 300 <100 079723 Order
Acclaim SEC-1000 LC Columns
1000 7µm Guard Column 4.6 33 - 082739 Order
1000 7µm HPLC Column 7.8 150 <150 079722 Order
1000 7µm HPLC Column 7.8 300 <300 079721 Order
1000 7µm HPLC Column 4.6 300 <100 079724 Order

About size exclusion chromatography

The mode of separation in Size Exclusion Chromatography (SEC) is considerably different from Reverse Phase (RP) and other traditional modes of chromatographic separation. Where other modes rely mostly on the interaction of the analytes with the stationary phase, SEC differentiates molecules based on the molecule size using columns packed with porous particles. The stationary phase is designed so to minimize—ideally completely remove—any interactions with the analytes. Any secondary interactions between the analyte and column particle could negatively affect retention.

Size exclusion chromatography for proteins and biologics

SEC is routinely used for the analysis of proteins and more specifically often to evaluate the aggregation profile of the protein. Aggregation is a degradation product and must be closely monitored and kept within acceptable limits. This is a Critical Quality Attribute (QCA) for monoclonal antibodies (mAbs) and other antibodies because it can cause an immunogenic response in the patient. Large antibody aggregates are sterically excluded from most pores and therefor flow quickly flow through the column. Smaller proteins spend more time diffusing inside of the particle pores and elute later from the column. The smallest proteins, such as antibody fragments can enter all pores and elute last.

Size exclusion chromatography for water soluble polymers

SEC is also used for water soluble polymers and oligomers. Water soluble polymers are a highly varied family of products developed and used by medical, cosmetic, food, oil, agriculture, environmental, and many other industries. Examples of water soluble polymers include polyethylene glycols (PEGs), polyvinyl alcohols, polyvinyl pyrrolidones, dextrans, polyacrylic acids (PAA), polyacrylamides (PAM), polyamines and polyethyleneimines, quaternary ammonium polymers, and polyvinylpyrrolidone.

How SEC works and why pore size matters

The differential separation is then achieved because of the differential rates of diffusion for different molecules through the pores of the stationary phase. The rate of diffusion is dependent on the analyte’s size, or more specifically, the hydrodynamic radius. This is very closely linked to the mass of the analyte. Larger analytes are sterically excluded from an increased number of pores compared with a smaller analyte and so they pass through the column at a quicker rate with a lower retention time. This is the opposite to what you typically expect for analysis of larger analytes with reversed phase (RP) or hydrophobic Interaction chromatography (HIC) modes. There is a point at which larger analytes will be sterically hindered from all pores and elute in the void volume.

Similarly, there is a point at which smaller molecules will be able to diffuse into all available pores and the resolution power is also lost. This is also quite different to typical chromatographic modes, for example RP, where the retention will increase as the carbon chain length increases over a much wider range (until the molecule become too big for the pore size etc.) Therefore, the pore size is the critical factor that controls the separation mechanism and its efficiency. As a result, SEC columns are manufactured with a variety of pore sizes, for a variety of different applications and analyte sizes. The overall pore volume in the column particles controls the retention time of each species. The pore size of the particle controls the relative separation of proteins.

Special notes about SEC and the HPLC system

Size exclusion chromatography encounters some challenges, because unlike other chromatographic techniques, SEC has no focusing of analytes at the head of the column. So, chromatographic quality is also heavily dependent on dispersion of the HPLC chromatography system. Small particles reduce diffusion times and allow for shorter columns and faster separations. However, small molecules are difficult to manufacture with sufficiently large pores and they are prone to clogging / fouling. Some size exclusion chromatography column particles can be prone to robustness issues, with chromatography system pressure spikes the particle pores can deform, or rust particulates from the system can block pores, reducing column longevity and robustness.

MAbPac SEC HPLC columns for monoclonal antibody, aggregate, and fractions separation

MAbPac SEC-1 columns are specifically designed for the optimal, high-resolution separation and characterization of monoclonal antibodies (mAbs), monomers, aggregates, and fragments.

  • Analysis of monoclonal antibodies (mAbs) and their aggregates
  • Analysis of mAb Fab and Fc fragments, even using high and low salt concentrations
  • Hydrophilic bonded layer for minimal non-desired interactions between the biomolecules and the stationary phase
  • Stable surface bonding leads to low column bleed and compatibility with MS, ELSD, and Corona Charged Aerosol Detection (CAD)
  • Separation range for globular proteins 10,000−1,000,000; exclusion limit for globular proteins >1,000,000
MAbPac SEC-1

MAbPac SEC 1 HPLC Columns

Particle size Description ID (mm) Length (mm) Cat. No.  
5µm Guard Column 4 50 074697 Order
5µm HPLC Column 2.1 150 088790  Order
5µm HPLC Column 2.1 300 088789 Order
5µm HPLC Column 4 150 075592 Order
5µm HPLC Column 4 300 074696 Order
5µm HPLC Column 7.8 300 088460 Order

BioBasic SEC HPLC columns for protein and peptide separation

  • Superior separation of various biologics including peptides and proteins.
  • Separates analytes over a wide molecular weight range
  • Long column life and high column efficiencies
  • Simple mechanism of interaction based on molecular size and shape
  • Ideal for sample clean-up; often used as the first step in isolation of a protein from a crude sample.
  • Straightforward method development, simple mobile phases
  • When used with standards for calibration, it is possible to determine the molecular mass of proteins.
  • These columns can also be used for water soluble polymers
BioBasic SEC

Which BioBasic size exclusion column to choose

This chart provides molecular weight ranges (KDaltons) for BioBasic SEC columns

Phase Pore Size Proteins Pullulans Polyethylene Oxides/Glycols
BioBasic SEC 60 60Å 0.1–6 0.3–6 0.1–4
BioBasic SEC 120 120Å 0.3–12 0.3–100 0.4–10
BioBasic SEC 300 300Å 1–500 20–>500 2–100
BioBasic SEC 1000 1000Å 20–4000 20–>1000 Not Recommended
Pore Size (Å) Description ID (mm) Length (mm) Cat. No.  
BioBasic SEC 60
60 Guard Column 7.8 30 73305-037821 Order
60 HPLC Column 7.8 150 73305-157846 Order
60 HPLC Column 7.8 300 73305-307846 Order
BioBasic SEC 120
120 Guard Column 7.8 30 73405-037821 Order
120 HPLC Column 7.8 150 73405-157846 Order
120 HPLC Column 7.8 300 73405-307846 Order
BioBasic SEC 300
300 Guard Column 7.8 30 73505-037821 Order
300 HPLC Column 7.8 150 73505-157846 Order
300 HPLC Column 7.8 300 73505-307846 Order
BioBasic SEC 1000
1000 Guard Column 7.8 30 73605-037821 Order
1000 HPLC Column 7.8 150 73605-157846 Order
1000 HPLC Column 7.8 300 73605-307846 Order

Acclaim SEC HPLC columns for water soluble polymers

  • Separation of water soluble polymers and oligomers
  • Water soluble polymers are a highly varied family of products including glycols, polyvinyl alcohols, polyvinyl pyrrolidones, dextrans, polyacrylic acids, and more.
  • The Acclaim SEC-1000 has a nominal pore size of 1000 Å for separating polymers and oligomers in the MW range of 1,000
    to 1,000,000 Dalton
  • The Acclaim SEC-300 has a nominal pore size of 300 Å for separating in the MW range of 100 to 50,000 Dalton  

pH range 2-12
Pore size 300, 1000
Particle size 5µm, 7µm
Temperature <60°C 

Which Acclaim SEC column to choose

  Acclaim SEC-300 Acclaim SEC-1000
Substrate Hydrophilic polymethacrylate resin Hydrophilic polymethacrylate resin
Particle shape Spherical Spherical
Particle size 5μm 7μm
Pore size 300Å 1000Å (multi-pore)
Separation range for PEO* 100–50,000 Daltons 1,000–1,000,000 Daltons
Exclusion limit for PEO* 50,000–150,00 Daltons 3,000,000–7,500,000 Daltons

*PEO = polyethylene oxides

Pore Size (Å) Particle size Description ID (mm) Length (mm) Sample Loading (µL) Cat. No.  
Acclaim SEC-300 LC Columns
300 5µm Guard Column 4.6 33 - 082740 Order
300 5µm HPLC Column 7.8 150 <150 079726 Order
300 5µm HPLC Column 7.8 300 <300 079725 Order
300 5µm HPLC Column 4.6 300 <100 079723 Order
Acclaim SEC-1000 LC Columns
1000 7µm Guard Column 4.6 33 - 082739 Order
1000 7µm HPLC Column 7.8 150 <150 079722 Order
1000 7µm HPLC Column 7.8 300 <300 079721 Order
1000 7µm HPLC Column 4.6 300 <100 079724 Order

About size exclusion chromatography

The mode of separation in Size Exclusion Chromatography (SEC) is considerably different from Reverse Phase (RP) and other traditional modes of chromatographic separation. Where other modes rely mostly on the interaction of the analytes with the stationary phase, SEC differentiates molecules based on the molecule size using columns packed with porous particles. The stationary phase is designed so to minimize—ideally completely remove—any interactions with the analytes. Any secondary interactions between the analyte and column particle could negatively affect retention.

Size exclusion chromatography for proteins and biologics

SEC is routinely used for the analysis of proteins and more specifically often to evaluate the aggregation profile of the protein. Aggregation is a degradation product and must be closely monitored and kept within acceptable limits. This is a Critical Quality Attribute (QCA) for monoclonal antibodies (mAbs) and other antibodies because it can cause an immunogenic response in the patient. Large antibody aggregates are sterically excluded from most pores and therefor flow quickly flow through the column. Smaller proteins spend more time diffusing inside of the particle pores and elute later from the column. The smallest proteins, such as antibody fragments can enter all pores and elute last.

Size exclusion chromatography for water soluble polymers

SEC is also used for water soluble polymers and oligomers. Water soluble polymers are a highly varied family of products developed and used by medical, cosmetic, food, oil, agriculture, environmental, and many other industries. Examples of water soluble polymers include polyethylene glycols (PEGs), polyvinyl alcohols, polyvinyl pyrrolidones, dextrans, polyacrylic acids (PAA), polyacrylamides (PAM), polyamines and polyethyleneimines, quaternary ammonium polymers, and polyvinylpyrrolidone.

How SEC works and why pore size matters

The differential separation is then achieved because of the differential rates of diffusion for different molecules through the pores of the stationary phase. The rate of diffusion is dependent on the analyte’s size, or more specifically, the hydrodynamic radius. This is very closely linked to the mass of the analyte. Larger analytes are sterically excluded from an increased number of pores compared with a smaller analyte and so they pass through the column at a quicker rate with a lower retention time. This is the opposite to what you typically expect for analysis of larger analytes with reversed phase (RP) or hydrophobic Interaction chromatography (HIC) modes. There is a point at which larger analytes will be sterically hindered from all pores and elute in the void volume.

Similarly, there is a point at which smaller molecules will be able to diffuse into all available pores and the resolution power is also lost. This is also quite different to typical chromatographic modes, for example RP, where the retention will increase as the carbon chain length increases over a much wider range (until the molecule become too big for the pore size etc.) Therefore, the pore size is the critical factor that controls the separation mechanism and its efficiency. As a result, SEC columns are manufactured with a variety of pore sizes, for a variety of different applications and analyte sizes. The overall pore volume in the column particles controls the retention time of each species. The pore size of the particle controls the relative separation of proteins.

Special notes about SEC and the HPLC system

Size exclusion chromatography encounters some challenges, because unlike other chromatographic techniques, SEC has no focusing of analytes at the head of the column. So, chromatographic quality is also heavily dependent on dispersion of the HPLC chromatography system. Small particles reduce diffusion times and allow for shorter columns and faster separations. However, small molecules are difficult to manufacture with sufficiently large pores and they are prone to clogging / fouling. Some size exclusion chromatography column particles can be prone to robustness issues, with chromatography system pressure spikes the particle pores can deform, or rust particulates from the system can block pores, reducing column longevity and robustness.

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