Biomedical Engineering Reference
In-Depth Information
Fig. 3.5.
Parameters of a signal peak obtained in chromatography. V
e
elution volume, t
e
elution
time (retention time), h height of the peak, W width of the peak at 0.5 h, a and b indicators of
peak symmetry
of the ligand to the carrier. Up to now, cyanogen bromide ac-
tivation of polysaccharide carriers is the most used method for
coupling, but since it has a relatively low chemical stability and is
limited to amino groups bearing molecules, other coupling reagents
should be taken into consideration. Table 3.6 gives a survey of cou-
pling reagents useful in preparing affinity media. Table 3.7 lists
reagents suitable for coupling ligands to spacer molecules, such as,
for example,
α
ω
,
-diamines as 1,6-diaminohexane or spermidine or
ω
ε
-aminocarbonic acids as 6-aminohexanoic acid (
-aminocaproic
acid).
Generally, sample application is done as described in Sect. 3.4.1.
There are no differences between batch and column application
and there are also no limits concerning volume as long as capacity
of the matrix is not exceeded. Binding buffer should be optimal
for biospecific interactions, i.e., pH, ionic strength, and composi-
tion should be optimized, and cofactors and structure-stabilizing
agents should be present. Do not exceed protein concentration
over 10 mg
/
ml and dilute sample or matrix if the affinity or ligand
density is too high.
Elution of the bound ligand can be done by competition with
the free (unbound) ligand or by partial denaturation of the pro-
