Conventional Calibration is a widely used GPC technique that employs a single concentration detector, typically a Refractive Index (RI) Detector or UV/VIS to obtain a relative molecular weight.
We have seen from the separation theory that in GPC/SEC molecules are separated according to their hydrodynamic volume and then characterized by a detector or series of detectors as they elute from the column. In Conventional Calibration, a relative molecular weight (MW) and molecular weight distribution (MWD) are determined from the measured retention volume (RV) by means of a calibration curve (log MW vs. RV), which must be set up with the aid of a number of standards of known molecular weight.
The process of conventional calibration is as follows:
- Inject a series of narrow polymer or protein standards of known molecular weight.
- Measure the retention volume (RV) of the resulting peak apex.
- Construct a calibration curve of Log(MW) vs. RV
Note:
Retention Volume is measured instead of Time to eliminate dependence on flow rate, and you must select standards that cover the entire Molecular Weight range of the sample to be analyzed.
Having gathered this calibration data, we now create a calibration curve to describe the relationship between retention volume and molecular weight; this is usually done by fitting the data to a polynomial:
Now when the unknown sample is analyzed, it is a simple process to estimate its molecular weight. Proteins are usually monodisperse in molecular weight, but polymers are not and typically polymer scientists are interested in at least 3 properties of the molecular weight distribution. These are called the Weight, Number and Z-Average and are defined as follows:
Where Mi is the molecular weight at the ith data point, and Ci is the concentration (or weight-fraction) at the ith data point.
The drawback with Conventional Calibration is that you may not have available standards of the same type as your sample. Remember, GPC/SEC separation is based on hydrodynamic volume; therefore, in attempting to determine molecular weight by conventional calibration we are making the assumption that the sample is of the same density as the standards. This is often not the case, and hence we often refer to the results from conventional calibration as relative molecular weight or 'Polystyrene Equivalent' molecular weight if the standards used are polystyrene. In other words, if the sample is not exactly the same type of polymer or protein that is used in the calibration the molecular weight obtained will be incorrect.
To overcome this problem, we can to construct a calibration curve where the y-axis is proportional to the hydrodynamic volume, instead of molecular weight. Fortunately, the addition of a viscometer detector, and utilization of the technique of Universal Calibration allows us to do precisely that. |
