Determining molecular weights for P(D,L)LA in ethyl acetate – an internal validation

Nils Lüttgert, Lisa Loxterkamp, Juliane Böttcher, Kate Monks; applications@knauer.net

KNAUER Wissenschaftliche Geräte GmbH, Hegauer Weg 38, 14163 Berlin

Collage: KNAUER

Summary

A method for the analysis of low molecular weight P(D,L)LA by GPC with respect to its number average molar mass (Mn) and weight average molar mass (Mw) was validated intralaboratory. To achieve this a KNAUER HPLC system was used in combination with a linear GPC column. The obtained molar masses were compared with the values from the absolute methods and deviations were discussed. The method was validated with respect to its repeatability and robustness to changes in flow rate, temperature, injection volume and sample concentration.

Introduction

Plastics are one of the most widely used most versatile materials in the 21st century. However, the majority of the world’s annual production of approximately 370 million tons of plastics is neither biodegradable nor produced from renewable raw materials.(1) In contrast, poly lactic acid (PLA) is one of the most promising plastics with sustainable properties. The physical properties of plastics are mostly determined by their molar mass distribution, with the parameters Mn, Mw and the PD being the most important. GPC is the method of choice for the determination of these values. When no absolute method is available, the two most common calibrations are conventional and universal calibrations. For a conventional calibration, calibration standards of the polymer to be analyzed, or at the least a polymer of high chemical similarity, are required. For a universal calibration, first published by Benoit(2), a universal calibration curve is created via the Mark-Houwink relationship, so that a wide variety of polymers can be analyzed, for example with a narrow polystyrene (PS) calibration. In 1998, S. Mori published the results of a multistage round-robin test in which PS standards were analysed by GPC using conventional calibration (CC) with PS under various framework conditions.(3) The relative interlaboratory standard deviation (%RSD) for Mn of more than 20 % was reduced by setting strict framework conditions for the analysis, such as injection volume, sample concentration, and sample size.

This shows that certain standards must be adhered to obtain reliable values when using GPC. Therefore, a method has been provided in this publication to analyse lower molecular weight P(D,L)LA using universal calibration. The method was developed according to ISO 13885-1 and its robustness and accuracy were investigated intralaboratory.(4) Ethyl acetate (EtOAc) was used as solvent, as it is significantly more environmentally friendly than the conventionally used solvents tetrahydrofuran (THF) and chloroform (CHCl3).

Sample Preparation

For calibration with PMMA, a ReadyCal-Kit in the molar mass range 800 Da - 2 200 kDa from PSS was used (12 standards). The calibration standards were dissolved in 1 ml EtOAc for one hour and then mixed with butylated hydroxytoluene (BHT) as a flow marker to a concentration of 1.5 mg/ml. The concentration of the calibration standard was 1.5 mg/ml. For the PLA samples, a 20 kDa PLA standard was used, for which Mn and Mw were specified in the certificate. For all samples, approximately 20 mg were weighed out and dissolved overnight (o.n.) in EtOAc to a concentration of 3 mg/ml. The following day, the samples were diluted to 1 ml with EtOAc and the flow marker BHT was added (1.5 mg/ml). All experiments were carried out by the same operator.

Results and Discussion

Initially, a calibration was made using PMMA, since polystyrene (PS) proved unsuitable in EtOAc due to interactions with the stationary phase (VTN0021). The two largest molar masses were excluded due to the peak shape, resulting in an 10-point universal calibration with a 5th degree fit function. The overlay of calibration function and the chromatograms is shown in Fig. 1. For universal calibration with PMMA in EtOAc, the following Mark-Houwink parameters were used: K = 21.1·105 dL/g, α = 0.64.(5) For PLA K = 15.8·105 dl/g and α = 0.78 were used.(6)

Fig. 1 10-point PMMA universal calibration in EtOAc by PSS (ReadyCal). Blue = green cup; Red = red cup; Light blue = white cup. Fit function of the 5th degree with deviations less than 5 %. R2 = 1.

Accuracy
Tab. 1 shows the molar masses determined using CC and UC. The comparison with the values of the absolute method 1H-NMR shows that the universal calibration provides much more accurate values. The values for Mn and Mw were taken from the certificate, with Mn determined by 1H-NMR. The method used to determine the Mw value was not specified. The comparison of the molar masses determined using CC versus determination using absolute methods has shown that CC has large deviations of more than 100 %. In contrast, UC showed good results with a deviation of 8 % for Mn and 4 % for Mw. Round robin tests have shown that GPC methods, in the simplest case of polystyrene in THF using CC, have a high uncertainty in their reproducibility (interlaboratory) of 13–16 % for Mn and 6–10 % for Mw.(3) For more complex polymers, deviations can be larger.(7) There is a tendency for the Mn value to show greater uncertainty than the Mw value.

Tab. 1 Comparison of the determined molar masses using PMMA UC and CC with the molar masses determined by 1H-NMR. *Average of 6-fold determination with (%)-deviation respective to the standard.

Repeatability and intermediate precision
To test repeatability, 6 injections of a 1 mg/ml concen­trated PLA solution were injected consecutively. The relative standard deviation was determined to ± 83 Da (0.44 %RSD) for Mn and ± 28 Da (0.13 %RSD) for Mw. Furthermore, the intermediate precision was tested. For this purpose, the sample preparation was repeated on three consecutive days and a triple determination was performed in each case. The relative standard deviation was ± 85 Da (0.46 %RSD) for Mn and ± 48 Da (0.22 %RSD) for Mw. Both values meet the requirements of ISO 13885-1, which specifies values lower than 3 % for Mn and 2 % for Mw.

Robustness
Several method parameters were varied to investigate their influence on the result of the molar mass deter­mination and to determine the respective standard deviation. First, the temperature was changed by ± 2 °C. Fig. 2 shows that the method is robust for a change of ± 2 °C since Mn and Mw do not change significantly and do not show a trend. The standard deviations were between 48 and 116 Da (0.6 %RSD).

Fig. 2 Mn (dots) and Mw (squares) values at 23-, 25-, and 27 °C. Triple determination with relative standard deviation. 1 mg/ml, 20 µl injection volume.

The flow rate was changed by ±0.2 ml/min. Fig. 3 shows that the method is robust even when the flow rate is changed by ±0.2 ml/min, since the % RSD is low, and no trend can be detected. The flow marker shows its strength here, as Mn and Mw are also very stable in this experiment when applying flow correction.

Fig. 3 Mn (dots) and Mw (squares) values at 0.8-, 1.0-, and 1.2 ml/min. Triple determination with relative standard deviation. 1 mg/ml, 20 µl injection volume.

For all experiments, 20 µl of PLA sample were injected. The aim was to investigate the influence of the injection volume on the molar mass determination. Fig. 4 shows the result. There is no significant effect on the molar mass determination in the range 10–30 µl injection volume. The %RSD is low and no trend can be identified.

Fig. 4 Mn (dots) and Mw (squares) values at 10-, 20-, and 30 µL injection volume. Triple determination with relative standard deviation. 1 mg/ml injection volume.

When determining high molar masses, concentration effects can reduce the hydrodynamic volume so that the molar mass is underestimated. This is also the reason why ISO 16014-3 specifies a sample concentration of 0.5 mg/ml for polymers with a molar mass with an order of magnitude of or greater than 106 Da, whereas sample concentrations of up to 5 mg/ml are not a problem when the Mw is less than 105 Da. Since the PLA standard for validation is in the magnitude of 104 Da no concentration effects should occur at an injection concentration of 1 mg/ml. If this were the case, the molar mass would have to increase with decreasing sample concentration. To test this, Mw and Mn were determined for different concentrations in the range 0.1–3 mg/ml. The results in Fig. 5 show that there is no trend towards higher molar masses at lower concentrations, this means that the method can be considered robust in the concentration range 0.1 to 3 mg/ml.

Conclusion

These experiments have shown that the determination of the Mn and Mw of low molecular weight P(D,L)LA is easily possible using a KNAUER HPLC system combined with a gel permeation chromatography (GPC)-column. The intralaboratory validation proves that the developed method is robust within the limits shown here with respect to changes in temperature, concentration, flow rate and injection volume. Furthermore, the repeatability of the molar mass determination as well as the intermediate precision of the sample preparation was verified. It was shown that the CC fails for this column/solvent/polymer combination, whereas the UC provides good results. The conventional calibration did not fulfil the specification because the different hydrodynamic radii of PLA and PMMA in EtOAc were not considered. The flow rate has a very strong influence on the molar mass determination and must be as constant as possible. Slight fluctuations affect the retention volume. In order to take fluctuations into account, a flow marker, in this case BHT, was used and is recommend as mandatory for all GPC/SEC applications.

Fig. 5 Mn (dots) and Mw (squares) values at 0.1-, 0.2-, 0.3-, 0.5-, 1.0-, 2.0-, and 3.0 mg/ml sample concentration. Triple determination with relative standard deviation. 20 µl injection volume.

Materials and Methods

Standards, Eluents and Samples

System configuration

References

[1] https://de.statista.com/statistik/daten/studie/167099/umfrage/weltproduktion-von-kunst­stoff-seit-1950/. Stand: 2019. 25.08.2021, 14:08 Uhr.

[2] Benoit, F., Grubisic, Z., Rempp, P.: Journal of Polymer Science 1967, B, 5, 753.

[3] S. Mori, International Journal of Polymer Analysis and Characterization: 1998, 4, 6, 531-546.

[4] ISO 13885-1: Gel permeation chromatography (GPC) – Part 1: Tetrahydrofuran as eluent, 2020.

[5] A. Eriksson, Acta Chem. Scand: 1953, 7, 623-642.

[6] X. Kaitian et al., Journal of Applied Polymer Science: 1996, 59, 561-563

[7] R. Bruessau, Macromol. Symp.: 1996, 110, 15-32.

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Application details