Molecular weight distribution of a broad polystyrene standard

Juliane Böttcher, Lisa Loxterkamp, Jan Wendrich, Kate Monks; applications@knauer.net

KNAUER Wissenschaftliche Geräte GmbH, Hegauer Weg 38, 14163 Berlin

Photo: Pixabay

Summary

Gel permeation chromatography (GPC) and size exclusion chromatography (SEC) describe the separation of molecules based only on their molecular size. Using an AZURA® GPC it is possible to run GPC/SEC applications with aqueous as well as organic solvents, when changing the system modules with the suitable compatibility.

Introduction

GPC and SEC are based on the same separation principle, size exclusion. GPC can characterize polymers with determining the molecular weight distribution and the refractive index. In former times the term SEC was used for aqueous applications with stationary phases, e.g. dextran. GPC on the other side was applied with organic solvents and for instance polystyrene stationary phases. Nowadays both definitions are used side by side for the same separation. The peculiarity of GPC and SEC is that analysis is performed over one column volume, which results in a negative partition coefficient (k) for the analytes. The complete separation is therefore taking place over the selective permeation range which depends on the pore and exclusion volume [1]. The analyte needs to be dissolved in the eluent during separation to determine the exact molecular weight distribution. Sorption or chemical interactions with the stationary phase or wetted parts are objectionable. GPC is a relative molecular weight determination, where the best alternative is using the direct standard calibration in which case the standard is the same polymer type as the sample [2]. This application gives an example of the mentioned appliance. A calibration with narrow polystyrene standards on a polystyrene stationary phase was performed. With this calibration a broad polystyrene standard was analyzed. The obtained results are compared to the given values of the certificate of the broad standard to illustrate that KNAUER AZURA devices are suitable for GPC/SEC implementation.

Results

The elugram in Fig. 1 shows an overlay of the RID trace of the broad standard and the determined calibration curve. Here it can be seen that the elution volume or the peak maximum of the broad standard lies within the calibration range. By using the ClarityChrom GPC/SEC extension it is easy to obtain the desired information about the molecular weight distribution of the analyzed samples. Fig. 2 exemplarily shows the distribution of the broad standard. The certificate of the broad standard provides the following values: Mw=248000 Da and Mn=143000 Da. The polydispersity is given with a value of PD=1.73 [Mw/Mn]. In comparison the calculated result with ClarityChrom are Mw=247163 Da, Mn=126765 Da and PD=1.95 [Mw/Mn]. The deviation of the calculated values compared to the certificate might be a result of the used column with mixed porosities resulting in a broader weight distribution. This column normally is used for screening purposes and did not provide the best resolution. In addition, the broad standard signal is not completely within the calibration range, which makes the deviation of Mn and PD plausible. Another effect could be the approximated K and α coefficients which are not exactly according the measurement conditions due to the different temperature. For exact coefficient determination a viscosity detector should be used.

Fig. 1 Polystyrene broad standard elugram (blue) with the calibration curve (black) in the background.

Fig. 2 Molecular weight distribution of the polystyrene broad standard.

Sample Preparations

A calibration in a range from 580 Da to 364 kDa using the EasiCal PS-2 calibration kit was prepared. Standards were prepared directly in the vial and dissolved in approximately 1.5 ml THF. 30 mg of the broad standard polystyrene were weighed in a volumentric flask and dissolved in 10 ml THF overnight. The used K and α Houwink coefficient were provided by the ClarityChrom software with K=14.1 [dl/g*105] and α=0.7 at 25 °C. The elugrams of the measured calibration standards are shown in Fig. 3 and Fig. 4.

Fig. 3 Polystyrene Standard PS-2 A.

Fig. 4 Polystyrene Standard PS-2 B.

Conclusion

As pumps are available for normal phase operation, aqueous/organic and aqueous mode KNAUER devices are perfectly suitable for this application.

Materials and Methods

Tab. 1 Method parameters

Tab. 2 Pump parameters

Tab. 3 System configuration

Tab. 4 Standards and samples

References

[1] Wu, C. Handbook of size exclusion chromatography. Marcel Dekker (1995).

[2] Teraoka, I. Polymer Solutions: An introduction to physical properties. John Wiley & Sons, Inc. (2002).

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Application details