Fractionation and DOSY NMR as Analytical Tools: From Model Polymers to a Technical Lignin

One key challenge hindering the valorization of lignin is its structural complexity. Artificial lignin-like materials provide a stepping stone between the simplicity of model compounds and the complexity of lignin. Here, we report an optimized synthesis of an all-G β-O-4 polymer 1 designed to model softwood lignin. After acetylation, the polymer Ac-1(V) was fractionated using a protocol that involved only volatile organic solvents, which left no insoluble residue. Using diffusion ordered spectroscopy NMR in combination with gel permeation chromatography, it was revealed that this fractionated material behaved like a flexible linear polymer in solution (average α > 0.5). Acetylated kraft lignin was subsequently processed using the same fractionation protocol. By comparison with the model polymer, we propose that the acetylated kraft lignin is composed of two classes of materials that exhibit contrasting physical properties. One is comparable to the acetylated all-G β-O-4 polymer Ac-1, and the second has a significantly different macromolecular structure.

achieve ca. 5-10% residual signal at 98% gradient strength (compared to 10% gradient strength) using the 1D DOSY experiment with the ledbpgp2s1d pulse sequence. Each pseudo-2D experiment consisted of series of 32 spectra acquired with 65536 data points. The gradient pulses were incremented from 10% to 98% with a linear ramp. The temperature was set and maintained at 295 K. Data sets were processed by Fourier transformation in F 2 , using line broadening of 10 Hz, followed by a baseline correction. The DOSY analysis was then performed in Bruker Dynamics Center 2.3. Manual peak picking was performed for each dataset and peak intensities were used to measure the signal decay. Error estimation of the fit was performed at the 95% confidence level. All samples were prepared by dissolving 60 mg of material in 0.7 mL of d 6 -DMSO. The samples were then sonicated for 30 mins at 35 o C and then filtered through a 0.45 µm PTFE syringe filter. All samples were allowed to thermally equilibrate prior to optimizing DOSY parameters (∆ and δ).

Continuous wave electron paramagnetic resonance spectroscopy
EPR spectra were obtained with a Bruker EMX 10/12 spectrometer equipped with an SHQE resonator at an operating frequency of ~9.76 GHz with 100 kHz modulation. Weighed lignin samples were measured in 4 mm OD quartz tubes (Wilmad) closed with rubber septa (Sigma-Aldrich). All spectra were recorded with a time constant and conversion time of 40.96 ms each and an attenuation of 38.0 dB (32 µW power) to avoid saturation. For quantitative analysis, spectra were recorded in triplicate using a 200 G (20 mT) field sweep centred at 3485 G with 1024 points resolution and a modulation amplitude of 2.0 G. A 100 µM solution of TEMPO in toluene was used as external standard for quantification and measured with identical parameters. Wider scans to detect broader signals were recorded for the crude and derivate and the later fractions using a 800 G field sweep centred at 3485 G with 2048 points resolution and a modulation amplitude of 2.0 G. Narrower scans to resolve potential additional structure on the radical signal were recorded for the earlier fractions S4 using a 90 G field sweep centred at 3485 G with 1024 points resolution and a modulation amplitude of 0.4 G. Uncertainties in the number of spins per mg sample were estimated from the respective weighing and volumetric errors and the corrected standard deviations of triplicate measurements propagated to spins per weight.

Synthesis of alkyl bromo-esters 3(I-VIII)
To a stirred solution of the bromoacetic acid (6) (1.20 eq.) in cyclohexane (10 mL/g of alcohol) was added the required alcohol (1.00 eq.) and p-toluenesulfonic acid monohydrate (0.01 eq.). The mixture was heated at reflux under Dean-Stark conditions until the reaction was complete (as judged by 1.00 eq. of water removed). After cooling to room temperature, the crude reaction mixture was diluted with cyclohexane (1 vol. eq.) and washed with NaHCO 3 (sat. solution), brine, dried over MgSO 4 and concentrated in vacuo to give the required bromoester in sufficient purity that no further purification was carried out.
Quantities of reagents, reactants and solvents are specified for each reaction in the Supplementary Information.

Synthesis of Monomer Units 4 (I-VIII)
To a stirred solution of vanillin (1.00 eq.) and K 2 CO 3 (2.00 eq.) in acetone (10 mL/g) was added the bromoester (3(I)-3(VIII)). The mixture was heated at reflux until the reaction was complete (as judged by TLC). The reaction was then cooled, filtered through a pad of celite. and concentrated in vacuo to give the crude monomer (4(I)-4(VIII)). Quantities of reagents, reactants, solvents and the purification method employed are specified for each reaction in the Supplementary Information.

Synthesis of all G β-O-4 polymer 1 -(i) Aldol polymerization and (ii) Reduction
LDA Preparation: to a solution of diisopropylamine (1.3 eq.) in THF (3 mL/mmol) at -78 °C was added a solution of n-BuLi in hexanes (molarity determined by titration against diphenylacetic acid) (1.2 eq.) dropwise. The mixture was briefly warmed to 0 °C before cooling to -78 °C again prior to use.
(i) Aldol polymerization: To a flask containing monomer 4(I)-4(VIII) (1.0 eq.)* in THF (20 v/w) at -15 °C was added dropwise the LDA solution (see above). After addition, the reaction was stirred vigorously for 2 hours. The mixture was then quenched by addition of NH 4 Cl (sat. solution), diluted with brine and extracted with ethyl acetate (3x). The organic extracts were combined, washed with brine, dried with MgSO 4 and concentrated in vacuo to yield the crude polyester (5(I)-5(VIII)). * the monomer dried by azeotropic distillation using toluene (2x) immediately prior to use.
(ii) Reduction: To a solution of crude polyester (5(I)-5(VIII)) (1.00 eq.) in ethanol (10 mL/g) was added NaBH 4 (5.00 eq.) and the reaction was heated to 50 °C. Methanol (15.00 eq.) was added slowly and the mixture was left for 1 hour. The mixture was concentrated in vacuo to give a gum, re-dissolved in water (15 mL/g) and any alcohol produced during the reaction was extracted with ethyl acetate (3x). The aqueous layer was separated, acidified to pH ~2 using 6N HCl (caution: add slowly, very exothermic) which caused the reduced polymer to precipitate into a gum. The gum was collected and dried in a vacuum oven for 16 hours then taken up in acetone: methanol (9:1, ~10 mL/g) and added dropwise to diethyl ether (10x volume) to give an off-white precipitate of polymer 1(I-VIII).

Large Scale synthesis of the all G β-O-4 polymer
The synthesis of octyl bromoacetate (3(V)) and monomer unit 4(V) followed the identical procedures as reported above.
LDA Preparation: to a solution of diisopropylamine (9.42 g, 93 mmol, 1.5 eq.) in THF (280 mL) at -78 °C was added a solution of n-BuLi in hexanes (40 ml, 2.31 M (molarity determined by titration against diphenylacetic acid) 1.5 eq.) dropwise. The mixture was briefly warmed to 0 °C before cooling to -78 °C again prior to use.
(i) Aldol polymerization: To a flask containing monomer 4(V) (20.6 g, 64.0 mmol, 1 eq.)* in THF (250 mL) at -15 °C was added dropwise the LDA solution (see above). After addition, the reaction was stirred vigorously for 2 hours. The mixture was then quenched by addition of NH 4 Cl (sat. solution), diluted with brine and extracted with ethyl acetate (3x 200 mL). The organic extracts were combined, washed with brine, dried with MgSO 4 and concentrated in vacuo to yield the crude polyester (5(I)-5(VIII)). * the monomer was dried by azeotropic distillation using toluene (2x 200 mL) immediately prior to use.
(ii) Reduction: To a solution of crude polyester 5(V) (20.0 g, 62 mmol, 1.00 eq.) in ethanol (350 mL) was added NaBH 4 (12.1 g, 320 mmol, 5 eq.) and the reaction was heated to 50 °C. Methanol (30.8 g, 960 mmol, 15.0 eq.) was added slowly and the mixture was left for 1 hour. The mixture was concentrated in vacuo to give a gum, re-dissolved in water (15 mL/g) and any n-octanol produced during the reaction was extracted with ethyl acetate (3x 200 mL).* The aqueous layer was separated, acidified to pH ~2 using 6N HCl (caution: add slowly, very exothermic) which caused the reduced polymer to precipitate into a gum. The gum was collected and dried in a vacuum oven for 16 hours then taken up in acetone: methanol (9:1, ~10 mL/g) and added dropwise to diethyl ether (10x volumes, v/v) to give an off-white precipitate of polymer 1(V). *In some cases, the separation was inefficient. NaCl was added to the solution and the polymer precipitated out. The precipitate was collected by filtration and dissolved in the minimum amount of 6M NaOH. The resulting solution was neutralized by the addition of 6N HCl with vigorous stirring. The sticky gum obtained was then collected and dried in a vacuum oven for 16 hours then taken up in acetone: methanol S7 (9:1, ~10 mL/g) and added dropwise to diethyl ether (10 volumes, v/v) to give an off-white precipitate of polymer 1(V) (3.00 g, 25% over 2 steps).

Gel Permeation Chromatography
GPC analysis was carried out using a Shimadzu HPLC/GPC system equipped with a CBM-20A communication bus, DGU-20A degassing unit, LC-20AD pump, SIL-20A auto sampler, CTO-20A column oven and SPD 20A UV-Vis detector. Samples were analyzed using a Phenogel 5 µm 50A (300 x 7.8 mm) and Phenogel (5 µm 500A (300 x 7.8mm) columns connected in series and eluted with inhibitor free THF (1mL min -1 ) with a column oven temperature of 30 o C. The system was calibrated using polystyrene standards sourced from Polymer Standards Services (PSS) with M P values ranging from 266 Da to 12600 Da.
M n values were calculated using the equation: where M i is the molecular weight at given point i, according to a calibration and h is the signal intensity of a given log M measurement point i.
M w values were calculated using the equation:

GPC sample preparation
To a solution of polymer 1(I-VIII) (ca. 7 mg) in pyridine (0.5 mL) was added acetic anhydride (0.5 mL) and the solution was stirred for 16 hours. The mixture was concentrated in vacuo by azeotropic distillation with toluene (3x), ethanol (3x) and dichloromethane (3x).
The residue was dissolved in THF (1 mL) and filtered through a 0.45 µm PTFE syringe filter and submitted for analysis. Fractionation samples were already acetylated and so used directly from fractionation process. Samples were prepared by dissolving ca. 7 mg of S8 material in 1 mL of THF, passed through a 0.45 µm PTFE syringe filter and submitted for analysis. The GPC had been previously calibrated using polystyrene standards sourced from.

Model Polymer Acetylation
To a solution of polymer 1(V) in pyridine (5 mL g -1 , v/w) was added acetic anhydride (5 mL g -1 , v/w) and the solution was stirred for 16 hours. The mixture was concentrated in vacuo by azeotropic distillation with toluene (3x), ethanol (3x) and dichloromethane (3x).
The residue was dissolved in a minimum amount of DCM and added dropwise to diethyl ether (10 volumes, v/v). The precipitate was collected by filtration and dried in vacuo.

Kraft Lignin Acetylation
To a solution of Kraft lignin in pyridine (5 ml g -1 , v/w) was added acetic anhydride (5 mL g -1 , v/w) and the solution was stirred for 16 hours. The mixture was concentrated in vacuo by azeotropic distillation with toluene (3x), ethanol (3x) and dichloromethane (3x). The residue was dissolved in the minimum amount of DCM and added dropwise to diethyl ether (10 volumes, v/v). The precipitate was collected by filtration and dried in vacuo.

Fractionation of the acetylated all G β-O-4 polymer (Ac-1(V)) and lignin
To the purified acetylated polymer / lignin (~10 g), was added a solution of acetone (5%, v/v) in diethyl ether (100 vol/w) and the mixture was allowed to stir vigorously for 1 hour. The insoluble fraction was filtered off and dried under vacuum. The filtrate was concentrated and dried in vacuo. This process was repeated with 5% increments of acetone in diethyl ether solution until all material had dissolved. After the last fractionation step, all the fractions were then dried in vacuo for a further 8 hours before being weighed. For a schematic representation see Figure S3.

Aldol Polymerisation Reactions: Polyester 5(I)-5(VIII) analysis
All reactions were conducted on a 2 g scale to determine which monomer 4(I)-4(VIII) gave the highest degree of polymerisation (D. o. P.) in comparison to isolated polymer yield at the end of the polymer synthesis.  Table  1 of the manuscript.

Table S1
Synthesis yields and molecular weight characterisation of large scale synthetic batches of model polymer 1(V).

Synthetic Batch
Yield of

GPC Analysis of Model Polymer Ac-1(V) G-1
See Experimental Section of manuscript for an additional description of how the GPC analysis was carried out.  The observed polydispersity increases, eventually to values above the initial bulk material (F6 and F7) across all fractionation. This is thought to result from the significant tailing that can be seen in the GPC profiles (seen in all fractionations) of these fractions (Figure 2, manuscript and FigureS4 below). As tailing leads to a lower number average molecular weight (M n ), a higher PDI is expected for fractions with increased tailing. If this is the case, the number average molecular weight (M n ) will be more significantly affected leading to a higher PDI values. It is important to note however that the high molecular weight material expected to dissolve at later steps in the fractionation process, represent a small weight fraction of the initial bulk material (Table S2 & Figure S4). Any lower molecular weight contaminants that were not extracted in a previous, higher yielding step, are likely to significantly reduce the measured number average and generate higher PDI than the initial bulk material. S17 Figure S4. Overlay of GPC elution time profiles of fractionated Ac-1(V) (from fractionation G-1). Fractions have been colour coded. The bulk material is shown in black. Fraction profiles have been normalised and multiplied by the mass fraction (% recovered material) to illustrate the relative abundances across the molecular weight distribution.  were normalised so that the benzylic CH 2 end group integral (*) = 2. The α-proton integral was then taken as n (for fraction F1, G-1 n=5.23, see A(i)), and used to calculate the average number of linkages in the polymer chains. This value was then multiplied by the molecular weight of one unit (blue fragment) and summed with molecular weights of the two end groups.  Samples were prepared by dissolving 60 mg of material in 0.7 ml d 6 -DMSO. 1 H NMR spectra were processed in MestReNova 11.0.3 software package. Global spectral deconvolution (GSD) was used to measure the integrals of the peaks of interest. a) magnitude of the integral of the β-O-4 α proton relative to the benzylic CH 2 end group. M n is the number average molecular weight calculated from molecular weights of the fragments (see Figure S4) and the relative integrals of the β-O-4 α proton. *Fractions did not provide enough material for DOSY analysis at the required concentration.   (1), number average molecular weight (M n ) measured by GPC (2), polydispersity index (M w /M n ) measured by GPC (3) and number average molecular weight calculated from end group analysis (4) S22 Table S8. The values of the analysis of G-2 fractionation showing the weight average molecular weight (M w ) measured by GPC (1), number average molecular weight (M n ) measured by GPC (2), polydispersity index (M w /M n ) measured by GPC (3) and number average molecular weight calculated from end group analysis (4), PDI calculated using (M w (GPC)/M n (NMR)) (5). Integrations for the GPC were taken from where the UV response left to where it returned to the baseline.

Entry
Acetone    -2). The values were produced by taking average diffusion coefficient of peak picked signals for each fraction (See Figure S5).
Average log D values were produced by taking the log of the average diffusion coefficients. Concentration of samples was strictly controlled to 86 mg mL -1 . a samples that did not allow for an accurate concentration of solution to be made due to the abundance or properties of the material.

F13
n/a n/a n/a n/a -a a 1.22 -10.91

F14
n/a n/a n/a n/a -a a 1.08 -10.97