Size exclusion chromatography of polylactic acid in three different solvents

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

KNAUER Wissenschaftliche Geräte GmbH, Hegauer Weg 38, 14163 Berlin

Photo: pixabay

Summary

Several different polylactic acid (PLA) samples have been successfully examined with respect to their molar mass and polydispersity (PD) using a KNAUER HPLC system in combination with a gel permeation chromatography (GPC) column. Calibrations of polystyrene (PS) and polymethyl methacrylate (PMMA) in three different solvents, tetrahydrofuran (THF), chloroform and ethyl acetate (EtOAc) have been recorded. In this study a 20 kDa PLA standard and two real samples were evaluated using these calibrations and the results compared with one another. It could be shown that EtOAc is suitable as an environmentally friendly alternative for the analysis of low molecular weight P(D,L)LA samples.

Introduction

PLA is one of the most promising sustainable plastics. It can be produced on an industrial scale using simple, bacteria-driven, bio-based synthesis whilst also being biodegradable, meaning it could help solve problems such as long-term pollution of the environment by non-degradable plastics.(1) In addition, PLA can be used in a wide range of applications such as medical applications, as packaging material, for disposable tableware or in horticulture. Work is currently underway to make PLA more widely applicable and more recyclable through fibre reinforcement.(2) The macroscopic properties of a polymer, such as melting point, hardness and elasticity, are mainly determined by the number average molar mass (Mn), the weight average molar mass (Mw) and the polydispersity. The method of choice to determine these parameters is GPC. In this application, three methods are presented to determine the molecular weight of three different PLA standards. For this purpose, two calibrations in three different solvents, THF, CHCl3 and EtOAc, were investigated/recorded. In addition, two real samples were analysed: PLA filaments for 3D-printing and a PLA disposable drinking bottle.

Sample Preparation

For calibration with polystyrene, an EasiCal PS-1/2-Kit in the molar mass range 580 Da - 6 570 kDa from Agilent was used (17 points). For calibration with PMMA, a ReadyCal-Kit in the molar mass range 800 Da - 2 200 kDa from PSS was used (12 points). The calibration standards were dissolved in 1 ml of the corresponding solvent for one hour and then mixed with butylated hydroxytoluene (BHT) as a flow marker to a concentration of 1.5 mg/ ml. The number of calibration points for each solvent was adjusted to cover the optimal separation range of the column. The PLA standards were dissolved in the corresponding solvent over night at room temperature without stirring to a concentration of 1 mg/ml and then mixed with BHT as a flow marker to a concentration of 1.5 mg/ml. The PLA samples to be investigated were dissolved overnight at RT without stirring in the corresponding solvent to a target concentration of 3 mg/ml and then filtered over a 0.45 µm PTFE filter. BHT was added as a flow marker to a concentration of 1.5 mg/ml.

Results

A linear column was used for the analysis of the PLA samples. To calibrate a PS and a PMMA calibration were recorded in all three solvents. BHT was used as the flow marker. Fig. 1 shows the calibration with PS in chloroform while Fig. 2 displays the calibration with polymethyl methacrylate (PMMA). The calibration points were fit with a 5th degree function and showed deviations of less than 5 %. The following Mark-Houwink constants from the literature were used for the evaluation by means of universal calibration: K= 22.1 dl/g·105, α = 0.77 for PLA in CHCl3 and the respective Mn value were determined. (3) Tab. 1 shows the results compared to the Mn value of the certificate determined by 1H-NMR. There are significant deviations of over 20 % for the lower molecular weight polymers. It is striking that both PMMA and PS calibration show deviations in the same range. The calibrations have been checked against each other and provide good results.

Fig. 1 16-point PS calibration in chloroform by agilent (EasiCal) in the molar mass range of 1 230 Da – 2 703 kDa. Blue = PS-1(A); red = PS-1(B); light blue = PS-2(A); gold = PS-2(B). Fit function of the 5th degree with deviations less than 5 %. R2 = 1.

Fig. 2 12-point PMMA calibration in chloroform by PSS (ReadyCal) in the molar mass range of 800 Da – 2 200 kDa. Blue = green cup; red = red cup; light blue = white cup. Fit function of the 5th degree with deviations less than 5 %. R2=1.

Fig. 3 PLA standards in chloroform. Blue ≈ 5 kDa, red ≈ 10 kDa, black ≈ 20 kDa. T = 25 °C. 1 mg/ml, injection volume = 20 ul. PS calibration shown.

The following Mark-Houwink constants were used for the analysis of PLA in EtOAc: K = 15.8 dl/g·105, α = 0.78.(4) Tab. 2 shows the results of the analysis of the 20 kDa PLA standard in EtOAc with a PMMA and PS calibration. The PS calibration shows strong negative deviations of up to -150 %. The PMMA calibration, on the other hand, delivers good results. The following Mark-Houwink constants were used for the analysis of PLA in THF: K = 17.4 dl/g·105, α = 0.74.(5) Tab. 3 shows the results of the PLA standard in THF. In this instance both the PS and PMMA calibration show good results.

Tab. 1 By means of PS- and PMMA calibration, determined Mn values of three PLA standards in CHCl3

Tab. 2 By means of PS- and PMMA calibration, determined Mn values of three PLA standards in EtOAc

Tab. 3 By means of PS- and PMMA calibration, determined Mn values of three PLA standards in THF

To test the performance of the method in chloroform and THF, two PLA filaments for use in 3D printing as well as one PLA-drinking bottle were analyzed. The chromatograms of the filaments differ greatly. Fig. 4 shows the overlay of filament A with a 10 kDa PLA standard. In addition to the RID signal, two UV signals with a wavelength of 245 and 280 nm are shown. The size ratios of the signals to one another match with the PLA standard, so that it can be assumed that it is pure PLA. The same pattern is observable in the chromatogram of the PLA-drinking bottle, depicted in Fig. 5. In contrast, Fig. 6 shows the chromatogram of filament B in THF. While the PLA standard shows no absorption at 280 nm in THF, a strong absorption signal at 280 nm of filament B can be seen. Furthermore, the ratio between the RID and 245 nm signal is too large in relation to the signal of the corresponding standard. In addition, the UV signal is strongly shifted in its retention time in comparison to the RID signal. As a result, it can be inferred that filament B has been exposed to another, possibly polymer-specific, additional substance like another polymer (blend) or macromolecule that absorbs UV in this region. Tab. 4 shows the determined molar masses of a PLA-drinking bottle as well as the filaments A and B. All of them have relatively similar molar masses and PD’s.

Fig. 4 Overlay of PLA 10 kDa standard with a PLA Filament A for 3-D Printing. Black & light blue = RID, dark blue & green = 245 nm, red & gold = 280 nm. 3 mg/ml in THF, 20 ul injection volume.

Fig. 5 Overlay of PLA 10 kDa standard with a PLA driniking bottle. Black & light blue = RID, dark blue & green = 245 nm, red & gold = 280 nm.

Fig. 6 PLA Filament B for 3-D Printing. Black = RID, dark blue = 245 nm, red = 280 nm. 3 mg/ml in THF, 20 ul injection volume.

Tab. 4 Determined molar masses of PLA samples in THF using PS calibration

Discussion

In 1998, S. Mori published the results of a multistage round-robin test in which PS standards were analyzed using GPC and a conventional calibration with PS under various framework conditions.(6) In compliance with strict specifications regarding injection volume, sample concentration, calibration kit and baseline determination, the relative standard deviation for Mn was 13–16 %. In this work a universal calibration was applied, for which Mark-Houwink constants from the literature were used. Taking this additional uncertainty into account, deviations in the range of 20 % compared to the absolute method can be regarded as acceptable. The extreme deviations of polystyrene in ethyl acetate were investigated by analyzing further narrow PS standards. There was a clear systematic deviation as shown in Tab. 4. The smaller the molar mass of the PS standard, the greater the underestimation. This result clearly indicates an interaction of PS with the stationary phase (StyDiViBe).

Tab. 5 Four different PS Narrow Standards evaluated with universal PMMA Calibration in EtOAc

Conclusion

Considering the relatively high uncertainty of the universal calibration, it could be shown that a low molecular weight PLA standard can be analyzed in three different solvents using two different calibrations. Due to interactions with the stationary phase, polystyrene in EtOAc is not suitable when using a polystyrene GPC column. This could be shown due to systematic deviations of several PS standards. The analysis of two PLA filaments with a KNAUER HPLC system in combination with a GPC column has shown that real samples can be analyzed quickly and easily using this suggested method. When using a Diode-Array Detector (DAD), additional information about the sample composition can be obtained. Particularly noteworthy is the possibility of analyzing PLA in EtOAc, as EtOAc is significantly more environmentally friendly when compared to CHCl3 and THF.

Materials and Methods

Standards, Eluents and Samples

System configuration

References

[1] Singhvi M. S., Zinjarde S. S., Gokhale D. V., Journal of Applied Microbiology: 2019, 127, 1612-1626.

[2] Pressemitteilung: Recyclingfähiger, faserverstärkter Werkstoff aus 100 Prozent biobasierter Polymilchsäure – Fraunhofer IAP; abgerufen: 27.07.2021, 12:12 Uhr.

[3] R. Köhn, Z. Pan, J. Sun, C. Liang, Catalysis Commu­nications: 2003, Vol. 4, Issue 1, 33-37.

[4] Xu K., Kozluca A., Denkbas E.B., Piskin E., Journal of Applied Polymer Science: 1996, 1, 59(3), 561-563.

[5] Dorgan J.R., Janzen J., Knauss D.M., Hait S.B., Limoges B.R., Hutchinson M.H, J. Polym. Sci. B Polym. Phys.: 2005, 43, 3100-3111.

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

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Application details