SEC Calibration and Data Handling

For SEC a calibration must be obtained by running polymers of known molecular weight. The more usual practice is to run a series of calibrants of very narrow molecular weight distribution (often described as being monodisperse) and known peak molecular weight (Mp) and obtain a calibration of log

Mp versus elution volume. Alternatively, there are procedures for using broad molecular weight distribution calibrants, where the molecular weight distribution is well defined.

In practice, the most commonly used calibrants consist of narrow distribution polystyrenes, and a wide range of these is commercially available. A small range of other polymer types of narrow distribution calibrants is also available but usually with a restricted molecular weight range. The availability of well-defined broad molecular weight distribution calibrants is extremely limited.

The log Mp versus elution volume calibration is usually expressed by an equation which can be a simple linear expression or a polynomial. The software will frequently allow an excellent fit by using a high order polynomial but it is questionable whether an order of three should ever be exceeded. In practice, reasonable good fits are normally obtained using first, second or third order curves but even for third order fits, the potential for variation at low molecular weight, affecting the high molecular weight end of the distribution, is intrinsically present.

The calculation of molecular weight averages and plotting molecular weight distributions from a sample chromatogram and calibration is complex and the use of laboratory computers was introduced early to SEC. However, automatic data handling has often led to inappropriate manipulation of the data and care is always required. The whole chromato-

Retention volume

Figure 3 Chromatograms of four solutions containing 10 narrow molecular weight distribution polystyrene calibrants.

gram envelope for the polymer is used in the calculations and any minor variation in baseline placement or integration limits at the extreme ends of the distribution is likely to have significant effects on one of the calculated molecular weight averages.

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