Determination of the Accessible Hydroxyl Groups in Cellulose by Using

Phosphitylation and 31P NMR Spectroscopy

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

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 31P NMR

31P NMR

Background

Phosphitylation of cellulosic sample for 31P NMR analysis:

R O H Cl PO

OP

O

OOR+ + HCl

2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane

D.S. Argyropoulos, J. Wood Chem. Technol. 14 (1994), pp. 65–82.

Experimental Setup

Schlenk line with four ports (parallel experiments)

Distillation apparatus for tetrahydrofuran (THF)

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

Cellulose sample was transferred to the Schlenk flask under constantargon purge

Schlenk flasks were dried by usinga heating gun and a vacuum pump (cycle repeated 3 times with argonfillings)

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

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)

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)

This aliquot was then analyzed with quantitative 31P NMR

NMR tube contained known amount of relaxation agent (chromium acetylacetonate) dissolved in CDCl3

Sample for analysis

110115120125130135140145150155160165170175180 ppm

0.3

2

1.0

0

3.2

4Phospitylation reagent

I.S. (Guaiacol)

H2O

Typical 31P NMR Spectrum

The consumption of P(II) is directly proportional for the reactive hydroxyls in the cellulosic surface

Reaction wasfollowed bymonitoring the decreaseon this signal

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

5 30 180 1200

Time (min)

Rea

ctiv

e H

yd

rox

yls

(m

mo

l/g)

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 31P NMR analysis

Ovendry Samples

Theoretically, the maximum amount of reactive hydroxyls in 1 gram of celluloseis 18.54 mmol

30000 rev.

Control

5000 rev.

0

0.5

1

1.5

2

2.5

3

3.5

4

5 30 180 1200

Time (min)

Rea

ctiv

e H

yd

rox

yls

(m

mo

l/g)

Control refers to unbeaten sample

Airdry Samples30000 rev.

5000 rev.

Control

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

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5 30 180 1200

Time (min)

Rea

ctiv

e H

yd

rox

yls

(m

mo

l/g)

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

5 30 180 1200 5760

Time (min)

Rea

ctiv

e h

ydro

xyls

(m

mo

l/g)

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%

Controls 5000 rev.

30000 rev.100 mg of sample

300 mg of sample

0

0.5

1

1.5

2

2.5

3

3.5

5 30 180 1200 5760

Time (min)

Rea

ctiv

e H

ydro

xyls

(m

mo

l/g

)

0

0.5

1

1.5

2

2.5

3

3.5

5 30 180 1200 5760

Time (min)

Rea

ctiv

e H

ydro

xyls

(m

mo

l/g

)

0

0.5

1

1.5

22.5

3

3.5

4

4.5

5 30 180 1200 5760

Time (min)

Rea

ctiv

e H

ydro

xyls

(m

mo

l/g)

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

Moisture Contents

Sample Ovendry Airdry 69% RH

Control n.d. 7.80 11.0

5000 rev. n.d. 7.9 10.9

30000 rev. n.d. 9.7 14.2

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

Controls

RH 69%

Airdry

Ovendry

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5 30 180 1200 5760

Time (min)

Rea

ctiv

e h

ydro

xyls

(m

mo

l/g)

Sample with highest moisture showed highest reactivity

5000 Rev. Beating

RH 69%

Airdry

Ovendry

0

1

2

3

4

5

6

5 30 180 1200 5760

Time (min)

Rea

ctiv

e H

ydro

xyls

(m

mo

l/g

)

Beaten samples follow the same pathway although at RH 69% valuesare slightly higher than those with unbeaten samples

30000 Rev. Beating

RH 69%

Airdry

Ovendry

0

1

2

3

4

5

6

7

8

5 30 180 1200 5760

Time (min)

Rea

ctiv

e H

ydro

xyls

(m

mo

l/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.

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.