Gel Filtration or Size Exclusion Chromatography (S.E.C.)

BioScience 6 MB Agarose Macrobeads for affinity chromatography of cells; 6% agarose; 250-350µ; Note: CNBr activation is easy and inexpensive.

CNBr activated Sepharose 6 MB beads; 6% agarose (MacroBead); 200-300µ; only available in frozen, CNBr activated form.

SEM photo of a 1% LE Agarose gel at 22kX magnification.

Bead Composition

Agarose is generally recognized as the preferred matrix for bead composition. As a result, the highest quality biotechnology grade of agarose is used in all BioScience Agarose Beads. Specialty beads can, however, have one or more gelling components, which may or may not include agarose.

Aside from the gelling component, the optimum bead properties are chosen as a function of the intended bead application. For that reason, each of the major application methods are discussed, below, based on their optimal bead properties: i.e. affinity (AF), size exclusion (SE) and Ion exchange (IE) chromatography as well as magnetic bioseparations (MagSep).

Bead Size

The bead diameter determines the maximum size of biological substances which can be passed through the void space BETWEEN beads in a column. The pore size of an agarose bead determines the maximum size of a biological substances that can ENTER the bead. Agarose gel pore size depends on agarose concentration and is discussed, below, in more detail.

  • The For low pressure AF, SE, and IE chromatography of proteins: nucleic acids, or polysaccharides: 50 -150 µ
  • For low pressure AF chromatography of cells: 250-350 µ
  • For magnetic bioseparations: Size is not a critical parameter since there is no need to use a packed column to achieve the separation. For magnetic, agarose beads, however, a size between 20-90 µ has been most popular.
  • BioScience Bead size range: any interval over the size range from 20-350 µ. The most popular intervals are : 20 - 50 µ, 20 - 90 µ, 50-150 µ and 250-350 µ
  • The relation between bead size. bead volume and bead surface area: Considering each bead as a sphere:
    • Surface area =
    • Volume =

    The equations above illustrate that increasing the bead diameter by a factor of 2 will increase the bead surface area by a factor of 4 and the bead volume by a factor of 8. Despite the high porosity of agarose beads, surface area in relation to volume issues are important to consider in some applications.

Bead Size Polydispersity:

In #5, above, "bead diameter" was considered; but what do we mean by "bead diameter" when a bead sample typically contains a range of bead diameters? For example, the standard range of bead diameters for low pressure chromatography is 50 - 150µ. So what is the average bead diameter for such a sample? In most cases, a Poisson Distribution of bead sizes is assumed and the average bead diameter is approximated by adding the upper and lower ends of the bead size range and dividing by 2. A more rigorous characterization of polydispersity can be obtained by considering one or more of the following approaches:

  • The Mass (Volume) Median Diameter (DV 0.5) : the bead diameter which divides the beads into two equal halves. Thus 1/2 the mass is made up of beads smaller than this bead size and the other half by beads with diameters greater than this diameter.
  • The Sauter Mean Diameter ( D32) : the diameter of a droplet whose ratio of volume to surface area is the same as the whole bead population.

The DV 0.5 and D32 can be an aid to planning certain types of experiments but are not generally considered for chromatographic applications.

  • Chromatographic resolution:

    Resolution is inversely proportional to the average bead size. The smaller the average bead size, the better the resolution. But, back pressure, or resistance to fluid flow, through a column increases as average bead size decreases, so a bead size should be chosen which gives the required resolution in the shortest time.

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Bead Porosity & Gel Strength

  • Porosity:

    Agarose gel bead porosity is inversely proportional to the agarose concentration in the bead. The practical porosity for chromatography is measured as a function of the "exclusion limit": the molecular size of a polymer which will just barely be excluded from the gel pores and therefore remain in the interbead void space. For proteins and polysaccharides, the exclusion limit is expressed in daltons. For nucleic acids it's expressed in base pairs (bp). It should be remembered that polymer shape and size are functions of composition, pH, ionic stength and other factors. For example, globular polymers , like proteins, are smaller and more compact than linear polymers having the same MW; as a result, they will tend to diffuse faster in a porous gel matrix. For that reason, the exclusion limit of a given agarose bead for globular proteins of MW X Kd than it will for a linear polysaccharide of X Kd. Polymers (or particles) larger than the exclusion limit will remain in the void space during column transit. Polymers smaller than the exclusion limit will enter the pores of the bead as a function of their rate of diffusion. in relation to the flow rate through the column. A polymer solute's Kav (a number between 0 and 1.0) is a measure of how much the polymer entered the bead as opposed to remaining in the void space. The table below can be used as a guide for selecting the appropriate agarose porosity for your sample and application.

Selecting the Right Gel Concentration (porosity)

Agarose Concentration Protein Fractionation Range* (Kd) Polysaccharide Fractionation* (Kd) Nucleic Acid Exclusion Limit (bp)
1.0% 1,000 to 150,000 1,000 to 150,000 ( > 3,000)
2.0% 80 to 40,000 90 to 20,000 1340
4.0% 50 to 15,000 40 to 5,000 860
6.0% 10 to 5,000 10 to 1,000 180
  • Gel Strength:

    Agarose gel strength is directly proportional to agarose concentration. At a 1% concentration, a force of 1 Kg/cm2 is required to break the gel. At 4%, the agarose gel requires a 4 Kg/cm2. If the gel is crosslinked both the strength of the gel and it's resistance to denaturing conditions (freezing, urea, guanidine, DM50, Kl etc.) increase dramatically. The crosslinking does NOT change the porosity of the gel since the short crosslinks occur only in the agarose double helices and junction zones at the boundaries of the large gel pores. As a practical matter, the maximum flow rate for a given bead size will be directly proportional to it’s ability to resist compression (i.e. gel rigidity or strength).

BioScience Gel Filtration or Size Exclusion Chromatography (SEC) Beads

Note: bead size is 50-150 μ for all agarose bead products; also, agarose concentration is either 4% (A-4B) or 6% (A- 6B) as indicated.

Reference Brand Product & Prices Agarose Bead (%) BioScience Bead Equivalent (code) Settled Bead Volume Catalog Number BioScience Bead Price
BioGel A-20 M now discontinued 1 1% Agarose Bead A-1B-OH 100 ml A-0100-0100 $52.00
500 ml A-0100-0500 $205.00
1 L A-0100-1000 $370.00
No commercial counterpart 1 CL- 1% Agarose Bead; A-1B-CL-OH 100 ml A-0101-0100 $60.00
500 ml A-0101-0500 $240.00
1 L A-0101-1000 $430.00
Sigma# 2B-300; Sepharose® 2B;$99/100 ml 2 2% Agarose Bead A-2B-OH 100 ml A-0200-0100 $52.00
$302/500 ml 500 ml A-0200-0500 $205.00
$496/1 L 1 L A-0200-1000 $370.00
Sigma# CL-2B-300; Sepharose® CL-2B;$97/100 ml 2 CL- 2% Agarose Bead; A-2B-CL-OH 100 ml A-0201-0100 $60.00
$326/500 ml 500 ml A-0201-0500 $240.00
$515/1 L 1 L A-0201-1000 $430.00
Sigma# 4B-200; Sepharose® 4B;$99/100 ml 4 4% Agarose Bead A-4B-OH 100 ml A-0400-0100 $52.00
$302/500 ml 500 ml A-0400-0500 $205.00
$496/1 L 1 L A-0400-1000 $370.00
Sigma# CL-4B-200; Sepharose® 4B;$104/100 ml 4 CL- 4% Agarose Bead; A-4B-CL-OH 100 ml A-0401-0100 $60.00
$336/500 ml 500 ml A-0401-0500 $240.00
$543/1 L 1 L A-0401-1000 $430.00
Sigma# 6B-100; Sepharose® 6B;$100/100 ml 6 6% Agarose Bead A-6B-OH 100 ml A-0600-0100 $52.00
$326/500 ml 500 ml A-0600-0500 $205.00
$515/1 L 1 L A-0600-1000 $370.00
Sigma# CL-6B-100; Sepharose® CL-6B;$110/100 ml 6 CL-6% Agarose Bead
A-6B-CL-OH
100 ml A-0601-0100 $60.00
$369/500 ml 500 ml A-0601-0500 $240.00
$584/1 L 1 L A-0601-1000 $430.00
BioGel A-1.5 M; now discontinued 8 8% Agarose Bead
A-8B-OH
100 ml A-0800-0100 $52.00
500 ml A-0800-0500 $205.00
1 L A-0800-1000 $370.00
No crosslinked version of discontinued BioGel A-1.5 M 8 CL-8% Agarose Bead
A-8B-CL-OH
100 ml A-0801-0100 $60.00
500 ml A-0801-0500 $240.00
1 L A-0801-1000 $430.00
BioGel A- 0.5M now discontinued 10 10% Agarose Bead A-10B-OH 100 ml A-1000-0100 $52.00
500 ml A-1000-0500 $205.00
1 L A-1000-1000 $370.00
No crosslinked version of discontinued BioGel A-0.5 M 10 CL-10% Agarose Bead
A-10B-CL-OH
100 ml A-1001-0100 $72.00
500 ml A-1001-0500 $288.00
1 L A-1001-1000 $500.00

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