Sooner Scientific Catalog

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Separator Gel Filtration Products

Gel Filtration & Size Exclusion Chromatography

Separator Gel A and Separator Gel ACL are an extensive line of Agarose Beads available for use in gel filtration and size exclusion chromatography. Each Product is accompanied by a Certificate of Analysis covering all of the following physical and chemical properties: (a) Anionic moieties <0.2% (b) Methoxyl moieties <0.1% (c) 1% Gel strength >1,200 g/sq cm (d) Cytochrome C binding <8ug/ml. The gels are tested for bead geometry , size distribution and flow rate. Each bottle of gel contains 0.03% sodium azide as a preservative. See the equivalence chart for comparison with Pharmacia and Bio-Rad gel filtration products. We guarantee the Separator Gels to perform as well if not better than Pharmacia and Bio-Rad or we will refund your purchase. Bead size distribution options are standard (45u-150u), macro (250u-350u) and custom specifications.

Gel Filtration Equivalence Chart
Agarose Bead (%)	Brand Name	Supplier	Sooner Equivalent
1	Bio-Rad & Pharmacia	No Equivalent	Separator Gel A1
2	Sepharose 2B	Pharmacia	Separator Gel A2
2	BioGel A-50M	Bio-Rad	Separator Gel A2
4	Sepharose 4B	Pharmacia	Separator Gel A4
4	BioGel A-15M	Bio-Rad	Separator Gel A4
6	Sepharose 6B	Pharmacia	Separator Gel A6
6	BioGel A-5M	Bio-Rad	Separator Gel A6
8	BioGel A-1.5M	Bio-Rad	Separator Gel A8
10	BioGel A-0.5M	Bio-Rad	Separator Gel A10
12	Bio-Rad & Pharmacia	No Equivalent	Separator Gel A12
14	Bio-Rad & Pharmacia	No Equivalent	Separator Gel A14
16	Bio-Rad & Pharmacia	No Equivalent	Separator Gel A16

Table B: Agarose Bead Q.A. Specifications

Quality assurance (QA) is very important to Sooner Scientific, Inc. and, accordingly, we provide all of the following information on every product ordered -- plus any additional tests that are important to our customers.

Agarose Characterization:

1. Physical & chemical properties:

a. Anionic moieties < 0.2 %

b. Methoxyl moieties < 0.1 %

c. 1% Gel strength > 1,200 g / sq cm

d. Cytochrome C binding < 8 µg / ml

Bead characterization:

1. Agarose concentration

2. Crosslinking: (Standard = epichlorhydrin; others on request)

a. passes boiling water test

3. Bead size: >95% in specified range

4. Bead geometry: spherical

5. Flow rate: at appropriate head pressure, comparable or better than commercial counterparts

6. Slurry concentration: 80% settled beads; 20% supernatant

7. Preservative: 0.03% sodium azide

8. Other analyses & specifications: on request

Table C: Selecting the Right Gel Concentration

Agarose

Concentration

Protein

Fractionation

Range (MW in

daltons)

Polysaccharide

Fractionation

Range (MW in

daltons)

DNA

Fractionation

Range (b. p.)

80,000 to

40,000,000

90,000 to

20,000,000

1340

50,000 to

15,000,000

40,000 to

5,000,000

860

10,000 to

5,000,000

10,000 to

1,000,000

180

Technical Support

Gel Filtration (GF) or Size Exclusion Chromatography (SEC)

Introduction:

The term gel filtration (GF) refers to the ability of a porous gel to "filter" or separate a mixture of macromolecules according to their individual hydrated sizes. The hydrated size of a molecule is typically a function of its molecular weight, branching, the ambient pH, ionic strength, and some other factors (ambient detergents, urea or equivalents, chelators, or chaotropic agents). The latter parameters can, therefore, be chosen to facilitate certain molecular separations by GF. Scaled-up or preparative GF is often used as a molecular purification technique.

GF is done under low, medium, and high pressure conditions. Most agarose beads, however -- even after epichlorhydrin crosslinking -- can only be used under LOW PRESSURE. "Low Pressure" means < 2 PSI or 140cm of fluid "head pressure". Fluid head pressure refers to the distance from the liquid surface of the eluant reservoir to the point at which the eluant discharge becomes discontinuous (i.e. forms drops).

Agarose Gel:

For all of the following reasons, agarose gels have long been recognized as the ideal media for GF of most proteins, most polysaccharides and many types of DNA:

1. Forms high-dimensional strength gels (0.5-10+ %)

2. Controllable gel pore size

3. Relatively neutral, hydrophilic surfaces

4. Readily crosslinked and/or activated for ligand coupling

Controlling agarose gel porosity: The size of the gel pores is a continuous function of the agarose concentration. In the past, agarose GF media have arbitrarily limited to a certain discontinuous series of arbitrary concentrations (2, 4, 6, 8 & 10 %). Since there is little overlap between adjacent concentrations, this has sometimes created a selection dilemma. In addition, even for important applications, it has not previously been possible to optimize the agarose concentration -- and, hence, the gel porosity. For examples: 2.6 % instead of either 2 or 4 %; 3.40% instead of either 2 or 4%, etc.

The Gel Exclusion Limit: Regardless of the agarose concentration chosen, there is always a molecular size for proteins, polysaccharides and DNA, which will not enter the pores of the gel. This is the smallest molecule which is excluded from the gel and is therefore called the exclusion limit. All molecules larger than the exclusion limit, expressed in either daltons (for proteins and polysaccharides) or base pairs (for DNA) will also be excluded from the gel and will pass through the column in the "void volume" (defined below).

The Lower Limit of Resolution: In contrast, when very small molecules receive no significant steric resistance to diffusion through the gel matrix, they diffuse the longest distances and take the longest times to exit the gel. When there is no significant difference in steric retardation by the gel, for two small molecules, then they will not be resolved and must be run on a less porous column. The highest MW molecules where this is noted represent the "lower limit of resolution" for that particular gel.

Linear Operating Range: Between the gel exclusion limit and the lower limit of resolution, lies the linear operating range for the gel-- where semilog plots of molecular weight vs. elution volume (or retention time on the column) are linear and substances elute in descending order of their molecular weight (i.e. biggest first, smallest last).

To Select the Right Gel Concentration: See Table C to select an agarose gel concentration whose exclusion limit and lower limit of resolution are above and below your MW range of interest, respectively.

Controlling Bead Size: Another previous constraint was the relatively limited range of bead sizes that have typically been made available as cost-effective options. (typically about 50-170 µ and higher prices for larger or smaller ranges or bead size distributions). See Table D.

Selecting the Right type of Agarose: It may come as a surprise that all agarose is not the same because, in the past, agarose chromatography beads have always come in only one "flavor": "vanilla". Actually, there are many types of agar-bearing seaweed -- the raw material from which agarose is derived. The physical properties of the agarose are a function of both its raw material source and method of extraction and purification.

The most important agarose properties which can be controlled by type selection are listed below:

1. Molecular weight: In general, the higher the agarose MW, the higher its gel strength.

2. Presence of anionic moieties: Typically most textbook pictures of the agarose molecule (i.e. agarobiose repeating unit or "monomer") neglect to show the various anionic moieties which significantly alter its functionality in many applications. These moieties include: ester sulfates, pyruvates, as well as methoxyl groups.

All of these moieties are attached to the agarose molecule via hydroxyl groups which are thereby "used up" and cannot be activated for coupling to ligands as in affinity chromatograpy.

In addition, these moieties can interact with some substances thereby preventing a GF separation which is solely a function of molecular weight or size.

Key Agarose Molecular-Structure Issues

1. The agarobiose (i.e. two sugars) unit represents the repeating "monomer" in the agarose polymer. There are approximately 400 such repeating agarose units per polymer chain. The symbol "AP" indicates the point of correction to the rest of the agarose polymer.

2. The importance of the substituents on the 4 potentially free hydroxyl groups on each agarobiose unit:

If R = H, then the hydroxyl group is "free" to be derivatized (i.e. crosslinked or activated to attach ligands as in affinity chromatography).

If any R = CH3, then the coupling capacity of the agarose is reduced.

If any R = SO3 or pyruvate, then the coupling capacity is further reduced for either crosslinking or coupling activation (for affinity chromatography).

Basic Chromatography Theory

The volume of a packed bed of porous chromatography beads can be represented mathematically as follows:

Vt = Vo + Vi + Vp

Where:

Vt = The total volume occupied by both the beads and the inter-bead liquid.

Vo = The volume of inter-bead liquid in the "void space".

Vi = The volume within the beads--exclusive of the (agarose) polymer gel matrix.

Vp = The volume occupied by the agarose polymer fibers only.

For example, in going from a 2% agarose bead to a 4% agarose bead, both having the same bead size and chromatography bed size, we'd only expect to change Vp and reduce Vi by ~2%, but Vo and Vt would remain the same. On the other hand, by doubling the average bead size of a 2% agarose bead, we'd substantially increase Vo (hence flow rate) while Vp is constant and either Vi or Vt could be held constant -- but not both.

The Elution Volume (Ve): In order to standardize the behavior between different columns, bed packings, and even aliquots from the same population of beads, it is necessary to relate the elution volume (Ve) of any eluant to both the void volume (Vo) and total volume (Vt) of the column, where:

Ve = the liquid volume which must pass through the column to elute a given substance from it. In practice, Ve is the total eluted volume corresponding to 1/2 the height of the leading edge of the elution peak for that substance.

Kav = Ve - Vo /   Vt - Vo

Thus, the Kav expression subtracts out any variable influence in void volume between different columns or packed beds of the same beaded media.

If a substance is excluded from the gel it will elute with the void volume and Ve = Vo thereby making Kav = zero. In contrast, if the substance is too small to be significantly retarded by the gel pores, then that component's Ve will approach Vt and Kav will therefore approach one (1). The Kav for all other intermediate-sized substances will be some decimal fraction from 0 to 1.0.

In practice, the difference in Kav observed between two substances run on two different agarose gel concentrations accurately reflects their relative resolution.

The Influence of Pressure on Flow Rate

Over the low pressure range where agarose bead shape is not significantly compressed, Darcy's Law (for incompressible porous solids) provides a good guide to the parameters which influence observed flow rate:

Flow rate (ml.cm-2.h-1) = K (pressure* applied - exit pressure*) / column length (cm)

Where: K = a proportionality constant dependent on bead shape (ideally spherical) and void fraction (a function of bead size).

* pressure is typically measured in cm of water (difference between height of eluate reservoir and point at which eluate drips from the column--including post-column tubing).

Resolution: For both analytical and preparative applications, the objective is still to obtain adequate resolution between two or more sample constituents, where:

resolution (Rs) = distance between two separated zones / zone width

and the distance refers to a difference either in elution volume or time and the zone width is quantified in the same units.

Table D: Selecting the Right

Bead Size Distribution

U.S. Alternate Mesh Sizes	Mesh Opening	Typical Bead-Size Ranges
35	500 µ
40	425 µ
50	300 µ	Range for Macro Beads: 250 - 425 µ
60	250 µ	Range for Macro Beads: 250 - 425 µ
80	180 µ
100	150 µ		Another Range for Standard Beads: 45 - 180 µ
200	75 µ	A Common Range for Standard Beads: 45 - 150 µ
325	45 µ	A Common Range for Standard Beads: 45 - 150 µ
400	38 µ
NOTE: You may customize the bead-size distribution by choosing any two screens from the above list and ordering beads between the upper and lower screen mesh.

Bibliography

1. Andrews, P. "Estimation of Molecular Size and Molecular Weight of Biological Compounds by Gel Filtration", Glick,D.(Ed.), Methods of Biochemical Analysis, vol. 18, p 1-53, lnterscience Publishers, New York, London (1970).

2. Determann, H. "Gel Chromatography", 2nd Ed., Springer-Veriag Inc., New York (1969).

3. Fisher, L. " An Introduction to Gel Chromatography", American Elsevier, New York 1969).

4. Heftmann, E., (Ed.) "Chromatography", 2nd Ed., Reinhold Publishing Corp., New York (1967).

5. Hjerten, S. "Molecular Sieve Chromatography of Proteins" in New Techniques in Amino Acid, Peptide and Protein Analysis, Ann Arbor-Humphrey Sci. Publ. Inc., Ann Arbor, MI (1971).

6. Reiland, J. " Gel Filtration" , Methods in Enzymology, 22, 287-321, Academic Press, New York (1971).

7. Snyder, L.R, & Kirkland, J.J., "Introduction to Modern Liquid Chromatog.", Wiley-lnterscience, New York ( 1974).

This list acknowledges some of the early leaders in this field and is not intended to be comprehensive. A wealth of specific applications exists today and should be searched for under key words from suitable data.

Ordering/Price Information - Please see page 46 or the index.

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