Theory GPC/SEC - Viscotek / Malvern

Triple detection sets the standard for current GPC/SEC technology and has become the preferred method for characterizing natural and synthetic polymers and proteins. It employs a concentration detector, viscometer and light scattering detector acting in concert, with each detector providing complementary but different information:

The light scattering detector provides a direct measurement of absolute molecular weight and eliminates the need for column calibration.
The viscometer detector provides a direct measurement of intrinsic viscosity or molecular density, and allows the determination of molecular size, conformation and structure.
Concentration is measured with an RI or UV, and is necessary for the determination of both molecular weight and intrinsic viscosity.

Triple detection is able to accomplish all this - and more - without the need for lengthy column calibration. Simply running a single narrow standard will verify the instrument constants of the detectors as well as performing corrections for inter-detector shift and inter-detector peak broadening effects. The peak broadening calculations are critical to good multi-detector GPC/SEC and the calculations used in the OmniSEC software are the result of over 20 years experience of refinement.

Triple detection has a unique advantage in that it provides absolute molecular weight, molecular size (to less than 1 nm) and intrinsic viscosity, as well as information on conformation, branching and aggregation in a single GPC/SEC experiment. It is the only detection technique capable of giving this tremendously rich collection of data on-line in a continuous flow mode.

Molecular weight data can be combined with information such as branching and hydrodynamic radius to give a clear indication of the structure of the sample across its molecular weight distribution.

In Triple Detection molecular weight is determined directly by the light scattering detector.

In order to determine molecular weight, all commercial light scattering detectors utilize the Rayleigh equation, which simply states that the intensity of the scattered light is equal to an optical constant times the concentration times the molecular weight.

According to the Rayleigh Equation, the intensity of the scattered light must be measured at zero angle. Since such a measurement would require you to look directly into the incident beam, which is not possible, the intensity must be estimated at zero angle and there are essentially three different ways to do this:

Measure the scattering at a very low angle (LALS). At angles of 10 degrees or less, the error is minimized and can be ignored.
Measure the scattering at multiple angles and extrapolate to zero (MALS). This is accomplished by plotting the light scattering signal as a function of angle.

Both methods will provide accurate molecular weight data for small molecules, such as proteins, which exhibit no angular dependence. However, for these small molecules it is preferable to use a more sensitive measurement at 90° which is right-angle light scattering (RALS).

For large molecules LALS is the simples and most theoretically pure method of determining molecular weight. In the LALS approach, the intensity of the scattered light is measured at the lowest possible angle (7°), eliminating any errors due to angular dependence without resorting to data fitting or extrapolations.

In the multi-angle approach, data from 2 or more angles must be extrapolated to zero angle to estimate the molecular weight. When molecules are large, the extrapolation is non-linear and the data must be fitted to a selected non-linear function before being extrapolated to zero angle. The quality of this fit and extrapolation are critical to the accuracy of the estimated molecular weight. Data from multi-angle instruments are often quoted with an uncertainty (+/-), but this is only an estimation of the precision, and doesn’t give any clue to the real accuracy of the molecular weight data.

A common addition to the Triple detector system is the UV/VIS detector which acts as a supplemental concentration detector.

This Tetra Detector System yields another level of detail; since we have two concentration detectors, it is possible to determine molecular weights for co-polymers and protein conjugates, in addition to being able to quantify the amounts of the two co-monomers present across the distribution.

Furthermore, a UV detector enables us to calculate molecular weight when neither the concentration nor the dn/dc is known. This is often the case when dealing with proteins. With knowledge of the protein's extinction co-efficient it is possible to determine it's concentration via the UV detector.

Now that the concentration is known, it's possible to determine the dn/dc from the RI detector, and hence all their parameters are available to determine molecular weight.

Systems for GPC/SEC:

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GPC/SEC Theory: Triple detection

Systems for GPC/SEC: