Agarose PhysicalChemistry

Appendix B: Agarose Physical Chemistry

In This Chapter
Agarose Physical Chemistry
References208209
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Appendix B: Agarose Physical Chemistry

Agarose PhysicalChemistry

Agarose is a polysaccharide consisting of 1,3-linked
β-D-galacto­pyranose and 1,4-linked 3,6-anhydro-α-Lgalactopyranose (Figure 1). This basic agarobiose repeat
unit forms long chains with an average molecular massof 120,000 daltons, representing about 400 agarobiose
units (1). There are also charged groups present on thepolysaccharide, most notably pyruvate and sulfate. Theseresidues are responsible for many agarose properties,
and by careful selection of raw materials, these properties
can be controlled to meet specific needs.
OH OHOOOOOOHOH—EEO –mr

COO –Figure 3: Illustration of EEO. Anionic groups are fixed to the matrix andcannot move. Corresponding cations can move and sweep toward thecathode with their associated water of hydration.

The mechanism for gelation of agarose was first suggested
by Rees (4) and later demonstrated by Arnott, et al. (5). Itinvolves a shift from a random coil in solution to a double
helix in the initial stages of gelation, and then to bundlesof double helices in the final stage (Figure 4). The averagepore size varies with concentration and type of agarose,
but is typically 100 to 300 nm (6).
Gelation Mechanism

COOL
HEAT

Solution

COOL
HEAT

Gel I

Gel II

Figure 4: Gelation of agarose by formation of double helices connected inthree dimensions by zones of random coil configuration.

One of the most important factors contributing to thesuccess of agarose as an anticonvection medium is its
ability to exhibit high gel strength at low concentrations(≤6%). Gel strength is defined as the force, expressed ing/cm2, that must be applied to fracture an agarose gel of astandard concentration. As there are several test methods
used to measure gel strength, a direct comparison ofgel strength values between different manufacturers issometimes difficult.
The gel strength of a specific lot of agarose will decreaseover time because of the spontaneous hydrolysis of theagarose polysaccharide chains. This loss of gel strength
can be particularly noticeable after 5 years from themanufacturing date.

0.5
0.4
0.3
0.2
0.1
05

101520253 meq SO4 + pyruvate/100g

208+

SO4 –

SO4 –

Figure 1: Agarobiose – Basic repeating unit of agarose.

0Na+

Na+

n

Electroendosmosis (EEO) is a functional measure of
the number of these sulfate and pyruvate residues
present on the agarose polysaccharide (Figure 2).
Electroendosmosis (2,3) is a pheno­menon that occurs
during electrophoresis when the anticonvective medium(the agarose in this case) has a fixed negative charge. Inan electric field, the hydrated positive ions associated withthe fixed anionic groups in the agarose gel migrate toward
the cathode. Water is thus pulled along with the positive
ions, and migration of negative molecules such as DNA
is retarded (Figure 3). Electroendosmosis is quantitatedby subjecting a mixture of dextran and albumin toelectrophoresis, then visualizing them and measuring
their respective distances from the origin. The amount of
EEO (-mr) is calculated by dividing the migration distanceof the neutral dextran (OD) by the sum of the migration
distances of the dextran and the albumin (OD + OA): -mr= OD/(OD + OA).

SO4 –

SO4 –

Figure 2: Dependence of electroendosmosis on total anionic residues inagarose.

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Agents that disrupt hydrogen bond formation (chaotropicagents such as urea and potassium iodide) will decrease
the melting temperature, gelling temperature and gel
strength of agarose gels, or even inhibit the formation ofthe gel. Since some electrophoretic procedures call forthe incorporation of urea into the gel, it is important to beaware of this effect.
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1008060

SeaKem® LE Agarose
SeaPlaque® Agarose
SeaPrep® Agarose

4020001gelling temperature. Because these agarose types havea melting temperature of ≤65°C, it is possible to remelt
the agarose without melting the DNA double helix. Thus,
slices of SeaPlaque® GTG® or NuSieve® GTG® Agarosegels containing DNA can be melted and the nucleic acids
manipulated directly in the remelted agarose.
Reduction in Gelling Temp., °C

Temperature, °C

The energy needed to melt an agarose gel increases as
the gel concentration increases. The gelling temperatureof an agarose gel is also influenced by the gelconcentration (Figure 5). For this reason, gelling orremelting temperatures are expressed at a given agarose
concentration. This property is of practical value since it ispossible to vary gelling and melting parameters by using
lower or higher concentrations of agarose. The depen­denceof gelling and melting temperatures on concentration ismost pronounced at concentrations less than 1%.

Agarose PhysicalChemistry

Continued

302520Hydroxyethyl

15

Hydroxypropyl
Methyl10500.60.20.40.80
Degree of Substitution/Agarobiose Unit(Max. = 4)

AllylAcetylFigure 7: Effect of various substituents on agarose gelling temperature.

3 4 5 6Concentration, %2Figure 5: Effect of concentration on gelling (open symbols) and melting(solid symbols) temperatures of agaroses.

The agarose polysaccharide also contains uncharged
methyl groups. The extent of natural methylation is directly
propor­tional to the gelling temperature. Unexpectedly,
synthetically methylated agaroses have lower, rather
than higher, gelling temperatures (Figure 6, lower curve),
and the degree of synthetic methylation is inversely
proportional to the melting temperature. A subsequent
study demonstrated that a number of simple derivatives
have the same effect as synthetic methylation on gelling
temperature (Figure 7). Because of manufacturingconcerns, hydroxy­ethylation is preferred for large scale
derivatization of agarose.

References
1. Rochas, C., and Lahaye, M. (1989) Carbohydrate Polymers 10: 289-298.
2. Himenz, P.C. (1977) Principles of Colloid and Surface Chemistry, Marcel
Decker.
3. Adamson, A.W. (1976) Physical Chemistry of Surfaces, John Wiley &Sons.
4. Rees, D.A. (1972) Biochem. J. 126: 257-273.
5. Arnott, S., et al. (1974) J. Mol. Biol. 90: 269-284.
6. Griess, G.A., et al. (1989) Biopolymers 28: 1475-1484.

The major use of low gelling temperature agarose, such as
SeaPlaque® GTG® and NuSieve® GTG® Agarose, is to takeadvantage of the low melting point rather than the low
Gelling Temperature, ° C

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50

Natural4030

Synthetic20
10002468

10% Methoxyl
Figure 6: Dependence of agarose gelling temperature on natural and synthetic
methylation.

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