GPC/SEC Theory: Universal Calibration
In Conventional Calibration we discovered an inherent problem occurs when we analyze a sample which is chemically different than the standards used to calibrate the column. Unfortunately this is frequently the case, so to improve on Conventional Calibration we use an additional detector, the four-capillary differential viscometer detector, and a technique called Universal Calibration.
As previously mentioned in Conventional Calibration and the separation theory, the principle behind GPC/SEC is that macromolecules are separated based on their hydrodynamic radius or volume. If this is the case, to accurately calibrate a column we need to form a relationship between the hydrodynamic volume and the retention volume (Universal Calibration), as opposed to the molecular weight and retention volume (Conventional Calibration). The viscometer detector makes this possible because it directly measures intrinsic viscosity (IV), which is inversely proportional to the molecular density of the polymer or protein coil.
The product of molecular mass and intrinsic viscosity provides the hydrodynamic volume:
MW • IV = 5/2 • NA • Vh
Because a viscometer detector coupled with a concentration detector (RI or UV/VIS) can give us a direct measurement of intrinsic viscosity (IV), we can now create a calibration curve of Log(Mw * IV) vs. retention volume (RV).
The procedure is as follows:
- Inject a series of narrow standards of known molecular weight.
- Measure the retention volume of the resulting RI peak apex.
- Allow the software to calculate the IV of the peak from the viscometer detector signal.
- Construct a calibration curve of Log(MW • IV) vs. retention volume.
Now when we analyze our unknown sample, at each data point (RV) we can look up on the calibration curve to find Y = Log(MWi • IVi).
Since we measure the IVi from the viscometer we can calculate that MWi = 10Y/ IVi.
From here, we can use the equations described in Conventional Calibration to determine the Mn, Mw and Mz averages.
In addition to being able to determine true molecular weight distributions (Mn, Mw and Mz), molecular size (Rh and Vh) and intrinsic viscosity (IV), the viscometer detector also gives us important structural information on conformation, branching and aggregation. Intrinsic viscosity, which is the inverse density of the polymer coil in solution, represents a direct and sensitive structural parameter and, therefore, is an important parameter in the polymer industry.
The well-known Mark-Houwink plot:
IV = K • MWa
can be obtained by means of the double-logarithmic plot of intrinsic viscosity against MW. The Mark-Houwink plot is the central plot used for the analysis of polymer structure. It reflects structural changes in the polymer, such as branching and chain rigidity. The slope, described by the Mark-Houwink exponent "a" can vary between 0 for solid spheres and 2 for rod-shaped structures.
Changes in slope in the Mark-Houwink plot are used to identify when branching occurs.
Almost all the benefits drawn from the technique of Universal Calibration can also be obtained by the method of triple or tetra detection which enhances GPC/SEC further by using light scattering to measure directly the molecular weight avoiding the lengthy column calibration procedure and removing the effects of any non-SEC column interactions.
Systems for GPC/SEC:
The Viscotek TDAmax is a complete, temperature controlled, advanced, multidetector GPC/SEC system suitable for all macromolecular applications, particularly research. It consists of three unique and complementary components – The Triple or Tetra Detector Array (TDA), the GPCmax integrated solvent and sample delivery module and the OmniSEC software.
The Viscotek 270max is a modular advanced multi detector detector system that operates at ambient temperature. It is perfect for the routine full characterization of natural and synthetic polymers, copolymers and proteins.
The Viscotek RImax is a modular, conventional calibration system. It offers simple operation and full upgradeability to advanced detection. Designed for routine GPC/SEC and teaching purposes. Operates with the same powerful OmniSEC software as used in the advanced systems.
More information
Application communication: Universal calibration to study star-branched polystyrene It is shown in this application communication that the number of branches of a star polymer can be assessed by merely adding a viscometer detector to a conventional chromatography system.
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