Agarosa

Agarose Physical
Chemistry

Appendix B: Agarose Physical Chemistry

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

Agarose Physical
Chemistry

Agarose is a polysaccharide consisting of 1,3-linked
β-D-galactopyranose and 1,4-linked3,6-anhydro-α-Lgalactopyranose (Figure 1). This basic agarobiose repeat
unit forms long chains with an average molecular mass
of 120,000 daltons, representing about 400 agarobiose
units (1). There are also charged groups present on the
polysaccharide, most notably pyruvate and sulfate. These
residues are responsible for many agarose properties,
and by careful selection of raw materials, these properties
can becontrolled to meet specific needs.
OH OH

O
O
O

O
O

OH

OH

—

EEO –mr

COO –

Figure 3: Illustration of EEO. Anionic groups are fixed to the matrix and
cannot move. Corresponding cations can move and sweep toward the
cathode with their associated water of hydration.

The mechanism for gelation of agarose was first suggested
by Rees (4) and later demonstrated by Arnott, etal. (5). It
involves a shift from a random coil in solution to a double
helix in the initial stages of gelation, and then to bundles
of double helices in the final stage (Figure 4). The average
pore 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 ofagarose by formation of double helices connected in
three dimensions by zones of random coil configuration.

One of the most important factors contributing to the
success 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 in
g/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 of
gel strength values between different manufacturers is
sometimes difficult.
The gel strength of a specific lot of agarose will decrease
over time because of the spontaneous hydrolysis of the
agarose polysaccharide chains. This loss of gel strength
can be particularly noticeable after5 years from the
manufacturing date.

0.5
0.4
0.3
0.2
0.1
0

5

10

15

20

25

meq SO4 + pyruvate/100g

208

+

SO4 –

SO4 –

Figure 1: Agarobiose – Basic repeating unit of agarose.

0

Na+

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. In
an electric field, the hydrated positive ions associated with
the 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 DNAis retarded (Figure 3). Electroendosmosis is quantitated
by subjecting a mixture of dextran and albumin to
electrophoresis, then visualizing them and measuring
their respective distances from the origin. The amount of
EEO (-mr) is calculated by dividing the migration distance
of 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 in
agarose.

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Agents that disrupt hydrogen bond formation (chaotropic
agents such as urea and potassium iodide) will decrease
the melting temperature, gelling temperature and gel
strength of agarose gels, or even inhibit the formation of
the gel. Since some electrophoretic...