Handbook of Size Exclusion Chromatography and Related ...

homogeneous copolymer, that is, taking some kind of average dn/dc value
measured experimentally from a set of unfractionated sample aliquots. The
“accuracy” of such an assumption may then be checked by integrating the
concentration detector response and comparing this recovered mass with
the known injected mass. The greater the departure from homogeneity, the
greater should be the discrepancy of these two results. Unfortunately, the total
mass recovered may not be complete because of column retention of some
components: a further complication. Although some form of molar mass
distribution may be generated on this basis, its accurately reflecting the true nature
of the real molecular structure must remain uncertain.
There are certain classes of copolymers for which mass, size, and
compositional distributions (stoichiometry) may be obtained following separation
by using MALS in combination with both UV and DRI detectors. These
copolymers have a homopolymeric component that produces no UV signal, that is,
it has no absorption at the UV wavelength commonly used. Among them are socalled
conjugated proteins comprised of a protein to which has been attached
(either by natural or synthetic means) a polymer (conjugate) that has no
chromophoric components. Most common among these are polysaccharides
(producing “glycosylated proteins”) and poly(ethylene) glycol (producing
“pegylated proteins”). The stoichiometry of protein–protein complexes where
each protein constituent may be conjugated has been studied by many groups. Wen
et al. (21) and Kendrick et al. (22) illustrate the techniques most frequently applied
using UV, DRI, and MALS detectors, although there are some questions remaining
as to how the weighted dn/dc values are calculated. The Wen et al. paper provides
an interesting discussion of an iterative approach whereby the relative proportions
of the (possible) two conjugated proteins are derived iteratively. The special case
whereby the UV detector may be used adds some simplification to the Benoit and
Froelich (20) method that introduced the concept of an apparent molar mass that
varied with the solvent used. The UV detector adds additional information of help
in establishing the stoichiometry of the molar mass distribution expected to be
present even within an SEC-separated elution slice.
A particularly simple example associated with such conjugated structures
occurs when the “core” is a single protein monomer. Separation of the conjugate by
SEC should be by hydrodynamic size and this in turn depends only on the amount
of conjugate attached. Each component has its distinct value of dn/dc and the
MALS measurements are combined with both UV and DRI measurements of the
eluting sample. The UV signal at each elution volume yields the concentration of
the polypeptide (protein) in that elution. If 1p is the protein extinction coefficient
whose corresponding differential refractive index increment (for the solvent used)
is (dn=dc) p, the protein concentration cpi at elution slice i is simply UV =1p where
UV is the calibrated UV detector response. If (dn=dc) B is the differential refractive
index increment of the conjugate, the weighted differential refractive index
© 2004 by Marcel Dekker, Inc.