First candidates: Electrostatic problems

As mentioned above one of the major issues when performing DOSY-NMR experiments is the overlapping peaks. The diffusion coefficient of molecules is obtained through an attenuation of the NMR signal in the spectrum after an evolution period, so, if there are two or more overlapping signals in the spectrum, the diffusion coefficient obtained will be an average diffusion coefficient of the molecules that are showing the overlapping signals. In order to prevent this problem it is useful to choose candidates (figure 3.1) that show signals in different parts of the spectrum. Therefore, the first polymers selected to perform a well resolved DOSY experiment in the spectral dimension were an aromatic polymer like Poly (Styrene Sulfonate) (PSS) (signals shown between 6-8 ppm) that had already shown ability for SEC studies [1] and a polymer with an amine functional group such as Polyallylamine (Paa) (signals shown between

1-3 ppm) which their ¹H-NMR spectrums are shown in figure 3.2. Both of these polymers show additional signals due to the CH2 backbone of the polymer. However, these signals have no interest in the performed studies as the signals produced by the backbones of the two different polymers will appear on the same region of the spectrum and they will probably show overlapping between them.

Figure 3.1: Poly (allylamine) (Paa) (left) and Poly (Styrene Sulfonate) (PSS) (right) repeating units

Following the studies done previously by Joyce and Day [1], the diffusion modulating effects that are studied in this chapter are expected to be explained through the interactions between the molecules and the stationary phase, and how different are these interactions when the particles have different sizes. However, the possibility of the molecules to explore the stationary phase depends on the size of the pores of the stationary phase (see table 2.1) and the size of the molecules. Hence, to simplify as much as possible the first results, one of the candidates will fit into the pores but will still be big enough to show interaction with the stationary phase.

Therefore, should be strongly affected by the stationary phase and vary its diffusion coefficient, while the other candidate will not fit into the pores and the interaction with the stationary phase should be minimum, which should be shown by either no variation or a very slight variation in the diffusion coefficient. Therefore, the molecular weights of the chosen candidates differ significantly (PSS Molecular weight: 70,000 Da and Paa Molecular weight: 15,000 Da). In addition to the molecular weight, concentration and temperature, the diffusion coefficient of the polymer will also be determined by the shape of the molecule [109]. Since both of these polymers are charged when dissolved in water, a high ionic strength buffer solution was needed to be added to eliminate chain expansion effects due to the polyelectrolyte in solution [108, 109].

Figure 3.2: ¹H-NMR spectrum 0.1 % w/w of 70 kDa Poly (Styrene Sulfonate) (left) and 15 kDa Poly (allylamine) (Right) in D2O with 25mM NaCl buffer

The diffusion coefficient of each polymer was measured at 25°C through a DOSY-NMR experiment (figure 3.4) in a set of samples with 0.1 % w/w of polymer and a range of NaCl concentrations 25, 50, 75, 100 and 150 mM, the results are shown in figure 3.3.

Figure 3.3: Diffusion coefficients of separated 0.1 % w/w 15 kDa Poly (allylamine) (1.95 ppm) and 70 kDa Poly (Styrene Sulfonate) (7.48 ppm) polymers in presence of different

concentrations of NaCl in D2O

Looking at figure 3.3 the candidates seem to behave as it would be expected by two molecules of significant different sizes, because the smaller molecule moves faster than the bigger one which suggest that they could be good candidates to perform SEC studies. However, it can be observed that while Paa diffusion coefficient remains very similar at the different NaCl concentrations, PSS diffusion coefficient increases until the concentration is up to 75 mM. This is probably due to changes in the shape caused by intramolecular electrostatic forces, only when the concentration of salts is high enough the molecules are completely surrounded by counter ions that ensure a uniform shape of the molecules. Therefore, suitable conditions for the experiment are 0.1 % w/w of polymer and NaCl concentration above 75 mM in D2O. To make sure that the candidates are suitable for the study it is needed to see their diffusion coefficients when they are in a mixture to make sure that there is no entangling or interaction between them that will interfere with their diffusion. The behaviour should be very similar, possibly a bit slower due to the presence of both polymers and changes in the viscosity. For this reason, it is advisable to put an upper limit to the concentration of polymer used.

Figure 3.4: DOSY-NMR spectrum of 0.1 % w/w of 15 kDa Paa in 25 mM NaCl buffer in D2O, the protons next to the N appear at 3ppm, the proton from the ramification of each monomer

appear at 1.98ppm and the aliphatic protons from the main chain appear at 1.5 ppm

When the mixture of the two polymers was prepared, a white solid was formed in all the samples and only the Paa was observed in the NMR spectra. In order to know whether the two polymers precipitate because of the difference in their charges or only the PSS was in the formed solid, similar experiments were repeated with an excess of PSS. This time the same white solid was formed but PSS appeared now in the NMR spectra of the mixture. This result suggested that both polymers are soluble when they are free in D2O. However, when they are in a mixture with

a polymer that has a chain with a net charge of different sign both polymers form an aggregate that is not soluble in D2O. The results are in agreement with the work done with polyampholytes, that are polymers that carry positively as well as negatively charged groups, by Everaers et al.

[112] and Dobrynin et al. They discussed:

“The solubility of polyampholytes and the composition of solutions at finite concentration. For ordinary polyelectrolytes, which carry charges of only one sign, the water-solubility is mostly due to the gain in translational entropy of the counter-ions in the water phase. The polymers are dissolved in spite of their high electrostatic self-energies, which they minimize by adopting stretched conformations. In contrast, polyampholyte samples can be self-neutralizing, thus resembling mixtures of oppositely charged polyelectrolytes. One can, therefore, expect the formation and precipitation of neutral complexes at finite concentrations.”[113].

In the case of our studies the combination of two polyelectrolytes with a net charge of opposite sign in solution seem to reproduce the behaviour seen in polyampholytes where a neutral complex is form. In order to solve this issue higher concentrations of buffer were tried up to 3 M to see if the buffer could act as an electrostatic screen and if it was possible to prevent the polymers from interacting with each other. However, it was impossible to obtain a mixture where both of them remained in solution at the same time. For this reason these polymers are not suitable for proof of concept for SEC studies in a mixture together.