1. Determination of the Accessible Hydroxyl Groups in Cellulose by Using Phosphitylation and 31 P NMR Spectroscopy

2. Objectives  To probe the amount of accessible hydroxyls on cellulosic materials including cellulose nanocrystals  To develop a quantitative methodology to follow the surface development (accessible hydroxyls) of cellulose as a function of various treatments - Enzymatic - Chemical - Mechanical

3. Reactive Hydroxyl Groups on Cellulose  Working hypothesis: Phosphitylation reagent (2-chloro-4,4,5,5- tetramethyl-1,3,2 -dioxaphospholane) reacts with the hydroxyl groups on cellulosic surface. Therefore, the amount of reactive OH’s can be calculated from the consumption of phosphitylation reagent by using 31 P NMR

4. 31 P NMR Background  Phosphitylation of cellulosic sample for 31 P NMR analysis: ROH ClP O O P O O O R ++ HCl 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane D.S. Argyropoulos, J. Wood Chem. Technol. 14 (1994), pp. 65–82.

5. Experimental Setup  Schlenk line with four ports (parallel experiments)  Distillation apparatus for tetrahydrofuran (THF)

6. Experimental Setup  Cellulose sample (from Whatman #1) was suspended in 15 mL of freshly distilled THF, 5 mL of dry chloroform, 5 mL of dry pyridine and 0.03 mmoles of 4- (dimethylamino) pyridine (DMAP)  The mixture was kept in 50 mL Schlenk flask equipped with a magnetic stirrer, an Argon inlet and a septum for reagent addition via a steel syringe  600 microliters of 2-chloro-4,4,5,5-tetramethyl-1,3,2- dioxaphospholane [P(II)] was then added slowly via the septum under slight agitation

7. Cellulose sample was transferred to the Schlenk flask under constant argon purge Schlenk flasks were dried by using a heating gun and a vacuum pump (cycle repeated 3 times with argon fillings)

8.  Dry THF was injected to the Schlenk flasks (containing cellulose) via a steel syringe  Samples were stirred 10 minutes before adding the rest of the reagents

9. An addition of dry chloroform, Pyridine, DMAP and internal Standard (guaiacol) An addition of phosphitylation reagent (drop-wise)  In all experiments one flask with identical amount of reagents, but without cellulose, was kept on side (blank)  By comparing the amounts of remaining P(II) on reaction flasks to that on blank, possible overestimations were avoided (phosphitylation reagent decomposes slowly over time)

10.  Reaction kinetics were monitored by taking out an aliquot (600 microliters) of reaction mixture containing P(II) that has not reacted with sample.  Samples were let to settle down before sampling (cellulose falls to the bottom allowing clear sampling) sampling (cellulose falls to the bottom allowing clear sampling)  This aliquot was then analyzed with quantitative 31 P NMR  NMR tube contained known amount of relaxation agent (chromium acetylacetonate) dissolved in CDCl 3 Sample for analysis

11. Phospitylation reagent I.S. (Guaiacol) H2OH2O Typical 31 P NMR Spectrum The consumption of P(II) is directly proportional for the reactive hydroxyls in the cellulosic surface Reaction was followed by monitoring the decrease on this signal

12. Reactivity of OH Groups in Cellulose The reaction was first applied to airdry cellulose obtained from whatman #1 filter paper in order to find out the reaction time needed for the complete phosphitylation. Level off was observed after 30 minutes 0 0.5 1 1.5 2 2.5 3 3.5 4 5301801200 Time (min) Reactive Hydroxyls (mmol/g)

13. Beating of Cellulose  Pulp from filter paper were beaten for specified number of revolutions (5000 and 30000), respectively (PFI mill method, T 248 cm-85)  Beating fibrillates fibers and it is widely accepted method of simulating commercial refining practices  Refined samples were first homogenized and then airdried, ovendried or conditioned at 69% of relative humidity  Cellulose samples from different beating intensities with different moisture contents were then subjected for 31 P NMR analysis

14. Ovendry Samples Theoretically, the maximum amount of reactive hydroxyls in 1 gram of cellulose is 18.54 mmol 30000 rev. Control 5000 rev. 0 0.5 1 1.5 2 2.5 3 3.5 4 5301801200 Time (min) Reactive Hydroxyls (mmol/g) Control refers to unbeaten sample

15. Airdry Samples 30000 rev. 5000 rev. Control Control and 5000 rev. are almost identical whereas 30000 rev. showed increased reactivity

16. Relative Humidity 69% 30000 rev. 5000 rev. Control Increased moisture content opens up the cellulose matrix 0 1 2 3 4 5 6 7 8 53018012005760 Time (min) Reactive hydroxyls (mmol/g)

17. Scale-up Experiments  The methodology was validated by scaling up the sample size (100mg to 300mg)  Reactivity was found to be very similar with maximum deviation of 15%

18. Controls5000 rev. 30000 rev. 100 mg of sample 300 mg of sample 0 0.5 1 1.5 2 2.5 3 3.5 53018012005760 Time (min) Reactive Hydroxyls (mmol/g) 0 0.5 1 1.5 2 2.5 3 3.5 53018012005760 Time (min) Reactive Hydroxyls (mmol/g) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 53018012005760 Time (min) Reactive Hydroxyls (mmol/g)

19. Effect of Moisture Content  Individual samples from different pretreatments (beating) were tested to follow the accessibility changes on cellulosic matrix at different moisture levels  Increased moisture content was seen to have great influence toward the elevated reactivity of cellulosic hydroxyl groups

20. Moisture Contents SampleOvendryAirdry 69% RH Controln.d.7.8011.0 5000 rev. n.d.7.910.9 30000 rev. n.d.9.714.2  Determined by using electronic moisture analyzer (Sartorius)  Ovendry samples were assumed to have close 0% moisture content  69% RH samples were conditioned 48h at 23°C

21. Controls RH 69% Airdry Ovendry 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 53018012005760 Time (min) Reactive hydroxyls (mmol/g) Sample with highest moisture showed highest reactivity

22. 5000 Rev. Beating RH 69% Airdry Ovendry 0 1 2 3 4 5 6 53018012005760 Time (min) Reactive Hydroxyls (mmol/g) Beaten samples follow the same pathway although at RH 69% values are slightly higher than those with unbeaten samples

23. 30000 Rev. Beating RH 69% Airdry Ovendry 0 1 2 3 4 5 6 7 8 53018012005760 Time (min) Reactive Hydroxyls (mmol/g) The most refined samples turned out to be the most reactive ones (as expected). Furthermore the moisture content seems to have the greatest effect to the most refined samples.

24. Conclusions  Hydroxyl groups on the cellulose surface can be phosphitylated in heterogeneous system  Beating changed the reactivity of surface hydroxyls  Different reactivities were observed for dry and moist samples  Developed methodology will be further used to monitor changes in enzymatically treated cellulose samples.