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 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.

• 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 K_av (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.

  

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

  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).

Agarose is derivatized to adjust it's properties for various applications.

• Crosslinking for enhanced strength and stability:

  
  
  Agarose gels have generally been crosslinked with either epichlorhydrin or it’s analogs. As a result, proximal hydroxyl groups on adjacent strands of the double helices are covalently bridged via ether linkages and an isopropyl alcohol moiety. These crosslinks then hold the gel together under conditions that would otherwise weaken it: boiling water, freezing, high concentrations of urea, guanidine, KI or denaturing solvents like DMSO. There is NO practical reduction in the capacity of crosslinked gels to bind ligands because the “buried” hydroxyls which become crosslinked would not be available to large ligands anyway. Only those hydroxyls on the pore surfaces are available for preactivation and susequent Iigand binding.
  
  Note: Crosslinked agarose beads are highly recommended for shipment during the Winter months when they might freeze in transit.
  

• PreActivation for coupling to ligands and Affinity Chromatography:
  
  • Glyoxal derivative: For binding to ligand amine groups, a “glyoxal” moiety is attached to the agarose hydroxyl groups so that it’s terminal aldehyde can readilly form a Schiff Base with a ligand amine. The Schiff Base intermediate can then be reduced to form a stable secondary amine linkage to the ligand by using sodium cyanoborohydride as shown below, where AB = agarose bead and L = Iigand:
    
    
    
    
  • Aminoethyl derivative : For binding to ligand carboxyl groups, an aminoethyl moiety is covalently attached to the agarose hydroxyl groups so that it's terminal amine can readily form an amide derivative with a Iigand carboxyl group in the presence of a suitable carbodiimide. This reaction sequence is shown below, where AB = agarose bead and L = ligand:

    

  • For Immobilized Metal Ion Affinity Chromatography (IMAC):
    
    Bidentate ligands, like imino diacetic acid (IDA) and tridentate ligands like
    nitrilotriacetic acid (NTA), will readily bind divalent metal ions like those shown above.
    Such complexes have been shown to bind histidine sequences which can be inserted as markers for expression proteins. In this way, IMAC can serve as an important technique for the identification and purification of certain expression proteins. A cost-effective source of agarose bead derivatives for IMAC can be found by clicking HERE.
    
    
    
  • For Ion Exchange Chromatography : For cation exchange, one can choose between carboxymethyl agarose, naturally carboxylated polysaccharide beads ( like alginate), or naturally sulfated polysaccharides (like carrageenan). For anion and cation exhange, one can choose arginine-coupled agarose beads.
    
    
  • Magnetic or Hydrophobic Beads:
    
    See the appropriate section on this website.
  
  References:
  
  
  
  1. Porath, J.(1992) Protein Express. Purif. 3, 263-281.
  2. Porath, J., Carlsson, J., Olsson, I and Belfrage, G. (1975) Nature (London) 258,
     598-599.
  3. Hemdan, E. and Porath, J. (1985) J. Chromatog. 323, 255-265.

 

Special Properties Unique BioAffinity Options: Often, the incorporation of micon-sized particles in the agarose gel will impart special properties which cannot be achieved - cost effectively- through chemical derivatization. In addition, certain stereochemistries inherent in natural products are almost impossible to reproduce through chemical derivatization of the agarose gel. For these reasons, we encourage the use of micron-sized gel inclusions and/or special, functionalized polymers within the agarose gel as a means of achieving the desired functional properties for a specialty bead. The schematic diagram, below, illustrates how small particles can be randomly distributed throughout the gel. Because of the high porosity of the agarose gel, solutes and target substances can diffuse into the gel and interact with the functional surface of the microparticles. Often, more than one type of particle will need to be dispersed in order to achieve the right balance of properties : for example, magnetism and hydrophobicity. Also shown in the diagram, is how one or more specially functionalized polymers can be dispersed and immobilized within the agarose gel so as to provide a unique functionality : for example, a heparin-like affinity. Gels containing functionalized polymers can also contain one or more particles to obtain the desired overall functionality:for example, magnetic susceptability and heparin-like properties.

BioScience beads with microcrystalline starch in 4% agarose bead; mag: 100 X   BioScience beads with microcrystalline starch in 4% agarose bead; mag. 400X   BioScience enzymatically sulfated gel bead stained with toluidine blue; 180-350µ; mag 100X