SECTION V

PREPARATION, FRACTIONATION, AND PRELIMINARY
CHARACTERIZATION OF GYMNOSPERM LIGNIN SULFONATES

Introduction

The first step taken to initiate the investigation was to proceed
with the preparation of the gymnosperms lignin sulfonates (for prior work,
see ref. 5,6,7). In view of the importance in the pulp and paper industry,
Western Hemlock wood was chosen as the basis for the investigation.

Purification and Separation

By courtesy of the Weyerhaeuser Company in Everett, Washington, spent
sulfite liquor was prepared by pulping Western Hemlock in a laboratory
digester under mild conditions utilizing caletum base pulping liquor.
Mild conditions were used in order to secure a larger than usual proportion
of low molecular weight lignin sulfonates in the resultant calcium base
sulfite liquor.

This calcium base liquor was then treated in accordance with the
flow sheet given as Figure |; and then, more specifically, according to
the following procedure. The liquor was degassed, passed through Dowex
50-8X to remove metal ions and yield free acids, then neutralized with

. barium hydroxide. The barium sulfate precipitate was removed by centri-
fuging and the final material concentrated by evaporation.

A sample of this concentrated BaSSL was applied to a column contain-
ing Dowex 50W-X2 ion exchange resin to achieve separation of strong
electrolytes (lignin sulfonates) from weak electrolytes (acetic acid,
aldonic acids) and non-electrolytes especially including sugars. The

effluent was collected intermittently after having been examined by a


ABSORBANCE i
Azsonm

PEAK C

PEAK A

9-13

—_

EFFLUENT VOLUME

FIGURE 9,
SEPHADEX LH-20 SEPARATIONS OF SOME ALSME FRACTIONS IN METHANOL.

32


of rather simple models have been reported (11, 12). Earlier attempts,
however, to obtain useful data from sulfonated lignin models failed
because of either the poor volatility or instability at elevated tempera-
tures. :

The acetylated methyl sulfonates, however, were found to be volatile
and thermally stable enough to yield reasonable spectra. Although the
base peaks were quite low in several cases, exact molecular wetghts could
be obtained. In our studies we recorded the spectra of monomeric and
dimeric models. A characteristic fragmentation pattern is shown in Figure 7.
Cone|ustons

Sulfonatton studies with lignin model substances provided us with some
information about the principal reaction pathway of the chemical reaction
that occurs in the sulfite pulping of wood.

The highly desirable use of modern analytical tools in organic chem-
istry was made possible through a new transformation procedure for sul fona-
ted lignin-like materials, which involved acetylation and esterification.
This method allowed transformation of the lignin sulfonates into deriva-
tives without electrolytic behavior.

With the aid of such acetylated and esterified lignin sulfonate mode
compounds the analytical basis for NMR and Mass spectroscopy studies with

lignin sulfonates from spent sulfite liquor was created.

28


in higher molecular lignin sulfonate fractions with an accuracy never
accomp|fshed before, even by applying rather elaborate analytical tech-
niques. The NMR spectroscopic technique represents the only method known
tn lignin chemistry so far which allows us to distinguish between these
two types of hydroxy! groups.

The correlation between these two hydroxy! functions led to a classi-
fication of the fractions in four different groups. A similar classtfica-
tion in a somewhat different fashion could be made on the basis of the UY
spectra (see Section V).

Significant changes tn the structural makeup of the low molecular
weight fractions of the ALSME were found, The predominant component in
the first five fractions exhibits features characteristic for |ignan-type
structures. Since unextracted Western Hemlock was used for acid bisulfite
pulping the presence of sulfonated lignans would not be surprising. Another
explanation, however, seems to be that this dimer is formed by a vinyl-
type condensation of two monomers with conjugated double bonds. Based on
the assumption that coniferyl alcohol is the only precursor, our findings
can be interpreted as depicted in the reaction scheme in Figure 12,

Homocondensation of coniferyl alcohol forms a lignan-type dimer with
the structure that represents the main compound in fractions !-5. Further
condensation between the dimer and conifery! alcohol and subsequent ally!
type rearrangement of the conifery! alcoho! side chain to a vinyl-type
structure leads to a product with the spectral features abserved in frac-
tions 8 and 9.

Although there is considerable doubt about the validity of the pro-
posed condensation mechanism, this hypothesis could very well explain some

of the observed discrepancies of LOMOLS. Further experiments are necess~

ary to support this hypothesis.
37


The apparent capability of of the ALSME to polymerize under certain
conditions suggests that further studies on the polymerizability of
lignin sulfonates should be conducted, and upon the nature and possible

commerctal use of the resultant polymers.


% Starting
Material
Dissolved o~
401

~~

Totat
4 Solubles
(Precip.)

30+ , °

Total
Solubles a
(Extr.) 7
Prd
4 a
o7 ls
Solubles
(Precip.)

-&-- - -4--
- LS Solubles
(Extr.)

IS 20
% Water in Ethanol

FIGURE 22.

DISSOLUTION OF SSL SOLIDS AS A FUNCTION OF
THE WATER CONTENT OF ETHANOL.

66


By Oxygen Function: It is well known that condensation reactions
compete with sulfonation reactions (Scheme 3, Figure 15) (26). In the
sulfonation reaction, a hydroxy! group is substituted by a sulfonate group
whereas condensation means elimination of a molecule of water.

A balance of oxygen atoms per Cy unit for all fractions is able to
give hints for the location of condensed LS in the Sephadex diagram. For
this reason all oxygen atoms per Cy unit are added up (Scheme 4, Figure 16).

!f a sulfonate group has substituted an OH-group, it is counted as | oxygen,

o
The oxygens attached to the sulfur atom are not counted.

4

The MWL starting material had 3.92 O/Cy (see Table 3). The deviation
from this value for each of the 12 fractions has been calculated and plot-
ted in Figure t7. All oxygen values lower than 3.92 0/Cy Indicate
condensation (or other loss of water).

The shape of the curve in Figure 17 Indicates that condensed LS are
"located" in the high- as well as the low molecular weight material, whereas
the material right between both areas (fraction 6) indicates no condensation
at all. Interestingly, this fraction is also the fraction with the highest
So, content of all, Condensed LS are found mainly in fractions 2 and 3 and
9-11. The degree of deviation of oxygens from the starting material
(0 - 0.25 0/Cy) can be considered to be an expression of the degree of con-
densation. Thus, fractions 2 and 3 contain LS, where every 4. or 5. aromatic
ring is condensed onto the side chain of another unit (probably mainly fn
6-position). Both fractions comprise about 10% of the total Signin sul-
fonates.

By NMR-Spectroscopy: This method has been shown to be a powerful

instrument in the analysis of Iignin model compounds as well as lignins

47


FIGURE 14, ELEMENTAL AND FUNCTIONAL GROUP COMPOSITION OF MWL - ALSME.

0.8 4
PHENOLIC OH
(NMR)

0.6

°
nn
sng

PHENOLIC OH / Cy
°
nS

7 — os A De ee A
10 8 6 4 2 lo 68 6 4 2
Fraction Number Fraction Number

>
a
& we ee ee ee oe 1.0
ia L- ~ Starting Material :
~ 0.8 4 on 0.8 4
an : hd a «
o 5
~ 0.6 4 ‘ 2 0.6 4
” ~
By = By Theory
S 0.44 OCH, / Cy F 0.4 4 Of Forss
4
0.24 0.2 4 Cy /8 So,
eo an SUI SEn eps Sen nes bes nein.an ones nen pen oe
10 «8 6 4 2 10 8 6 4 2
Fraction Number Fraction Number

45


These nucleophiles are, therefore, the most probable ones to undergo
condensation with carbonium ions (Scheme 3, Figure 15), thus contributing
to the lowering of the methoxy! content of the Itgntn sulfonates. The
sulfur content of LS.is represented by a bell-shaped curve, with the cul-
mination point at the border between high~ and low molecular weight LS.
tn LMWLS the sulfonation products of conifery! alcohol have been found,
probably attached to oligomeric fragments of lignin through the 6-position
of the aromatic ring.

Sumaa

Milled wood lignin (MWL) of Western Hemlock has been mildly sulfonated
and analyzed by Sephadex G-25 chromatography, NMR spectroscopy and elemental
analysis. A comparison of SSL from pulping of wood chips was found to be
in good agreement with the sulfonated MWL in all essential parts, except
for the very low- and very high molecular weight range. Results from the
MWL are, therefore, believed to be representative also for the lignin from
wood.

An exact division between low- and high molecular weight LS is pro-
posed by employing Sephadex chromatography, solubility in ether and ele~
mental analysis. According to this division, MWL is composed of 30% LMWLS
and 70% high molecular weight particles.

Structural studies revealed the content of phenolic hydroxy!-,
methoxy!- and sulfonate-groups. The degree of condensation was estimated.
Certain unique structural configurations could be assigned to some of the

LMWLS. An explanation for the demethylation observed was proposed,

51


_

I

EFFLUENT VOLUME

4
@

E.VOL.

FIGURE 18.

ELUTION DIAGRAM OF ES ALSME DERIVATIVES AND THE REFRACTIONATION PATTERS OF SOME MER CLASSES.


ov

TABLE 3

EMPIRICAL FORMULAE OF ALSME DERIVATIVES OF MWL

Fraction CH3-CO CH3-CO Mol. W.. Oxyg.

Number Cc H o* OCHz $030CH3, phenolic aliphat. Ave.for C, =H Bal.

Original

Sample - 9.00 8.40 2.98 0.94 -- -- -- 193.4 11.2 3.919
| 9.00 7.80 3.09 0.45 0.35 0.81 0.75 279.5 14.9 3.889
2 9.00 7.28 2.73 0.58 0.39 0.63 0.73 272.9 14.3 3.696
3 9.00 8.59 3.15 0. 1.5** 0.40 0.50 0.80 265.9 14.1 3.703
4 9.00 7.09 2.77 0.65 0.46. 0.49 0.80 279.5 14.3 3.889
5 9.00 7.23 2.77 0.68 0.46 0.42 0.70 272.5 14.0 3.906
6 9.00 7.12 2.60 0.67 0.51 0.45 0.78 279.2 14.3 3.779
7 9.00 7.44 2.73 0.73 0.49 0.44 0.8! 282.4 14,9 3.950
8 9.00 7.22 2.71 0.66 0.48 0.41 0.82 278.0 14.3 3.854
9 9.00 7.26 2.56 0.76 0.48 0.37: 0.76 274.2 14.4 3.798
10 9.00 7.57 2.61 0.69 0.45 0.33 0.80 270.9 14.4 3.750
"Wl 9.00 7.50 2.73 0.7! 0.36 0.29 0.60 254.3 13.4 3.802
12 9.00 7.26 2.89 0.70 0.33 0.34 0.81 264.4 13.8 3.920

* Oxygen-determination by difference.

** After repeating the analysis for Fs, the OCH,-content is about 0.60 per Cy.


fractions D - K is found at 280 nm and in fractions L - P at 281-282 nm.
This very small difference in the location of the absorption maximum can
also be seen in the spectra of the fractions of sulfonated mil led-wood
lignin.

In neutral solution fractions A, B and C of both spent sulfite liquor
and milled wood lignin show an absorption minimum at 260-261 nm whereas
fractions D, E and G show a minimum at 258 - 259 nm. Fraction | shows a
minimum at 256 - 257 nm.

In alkaline solution, fractions A, B and C show a maximum at 282 -
283 nm, fractions D, E and F at 285 - 286 nm. Fractions G - K show maxima
in the region 287 - 293 nm with the maximum shifting towards longer wave-
lengths with increasing effluent volume (and probably decreasing molecular
weights). Fractions L - P show the absorption maximum at 298 - 299 nm.

A division of the fractions of Figure 2 into the four groups | - IV
can be done on basis of the shift of the absorption maximum near 280 nm
on alkalizing the solution as can be seen from Figure 3.

Figure 3 shows that fractions A - C of the spent sulfite |iquor
showed a very small red-shift of only | - 2 nm. Fractions D - F showed a
red-shift of 5 - 6 nm, fractions G - K showed a red-shift of 8 - 13 nm and
fractions L - P a shift of 16 - 17 nm.

Characterization by Thin Layer Chromatography

About every third 10 ml fraction of Figure 2 was further studied by
means of thin layer chromatography (TLC) on analytical silica gel G
("Merck") plates using 4:1 isopropanol-water as eluent. The spots were

visualized by illuminating with ultraviolet light.


[Fractions 1-13 correspond with fractions A-P in Section V in reverse
order]), 1.0 (fraction 8, 9), and 1.4 Cfractions 10, 11) were observed
(see Figure 10). [In the next higher fractions we found a continuous
increase of this ratio.

A similar classification in a somewhat different fashion could be
made on the basis of the UV-spectra (See Section V). These findings were
finally supported by the remaining NMR signals which could be assigned
to protons of defined side chain structures. The main component in the
group consisting of the lowest molecular fractions, fractions 1-5, (See

Section V and Figure 2) was Indicated but not established to be a dimer

of a lignan-type structure.

The next higher fractions (6, 7) represent mixtures of different
structural makeup.

The lignan-like compound was finally isolated over Sephadex LH-20
and purified by recrystallization. . Its melting point, its NMR-spectrum,
and its mass spectrum indicate that it is of dimeric structure. Its abso-
lute configuration so far has not yet been established.

In fractions 8, 9 a vinyl sulfonic acid type side chain structure
could be detected.

The overlapping and broadening of the signals found in the spectra
of the next higher fractions does not allow further studies on side chain
structures. However, we did find indications that condensation presumably
in 5«position increases with the molecular weight. The first indication
of condensation in this way is observed in fraction 9 and seems to
increase steadily in the higher fractions. It remains unknown whether
this condensation ts Induced secondarily by the sulfonation reaction, or

whether it is present in the original lignin in the plant.

33


So

TABLE 4

Extraction Conditions:
hours at room temperature.

10g CaSSL extracted with 500 ml. ethanol and ethanol:water mixtures for twelve

a b c d e f g

(g) % Aa #550 (g) fs ér
Starting Material 10.0 100.0 9.100 100.0 7.6 76. 76.
Ex 100 0.6 6.0 0.022 0.24 0.018 0.12 3.0
Ex 95 0.9 9.0 0.184 2.0 0.15 1.5 16.7
Ex 90 1.50 15.0 0.980 10.8 0.82 8.2 54.5
Ex 85 2.20 22.0 1.430 15.8 1.19 i) 54.5
Ex 80 3.20% 32.0* 1.430 15.8 1.19 11.9 37.2*
* Tarry residue
Precipitation (dissolved matter)
Starting Material 20.00 100.0 18.2 100.0 15.20 76.0 76.
95-S 4.48 22.5 2.40 13.3 2.02 10.2 45.
90-S 6.00 30.0 3.65 20.0 3.05 15.2 50.
80-S 8.20 41.0 6.40 35.0 5.30 26.5 64.

Column Explanation

weight (g) recovered.

% weight based on starting material.

average absorbance of samples.

% of total absorbance at 280 nm.

theoretical weight of absorbing material where a assumed constant at 12.0.
% weight of absorbing material (LS) based on star#iag material. 1!/b X(e X 100)
% weight of absorbing material (LS) based on recovered fraction.

aroancro


ABSTRACT

Low molecular weight lignin sulfonates have been separated in
purified form and characterized by physicochemical and chemical methods.
Their structures and reactions have been evaluated. Preliminary studies
on the feasibility of industrial scale separations have been performed.

Lignin sulfonates from the spent sulfite liquor of a mild acid
bisulfite cook of Western Hemlock (Tsuga heterophylla) were purified and
fractionated in Sephadex G-25 column chromatography. Samples with varying
molecular weights were analyzed using a new method which aided the elim-
ination of the polydisperse nature of the material under investigation.
This method which involved acetylation of hydroxy!-groups and esterification
of sulfonate-groups was widely developed in the course of this study using
model compounds.

Complete elemental and functional group compositions were established
for lignin sulfonates from a spent sulfite |iquor and compared to those
from milled wood lignin preparation, allowed an estimate of the
degree of sulfonation, condensation and demethylation as well.

Extended separation studies indicated the low molecular weight lignin
sulfonates to be the reaction product of a difunctional vinyl-type poly-
merization, thus accounting for the widely different properties as compared
to their higher molecular weight counterparts (two- versus three-
dimensional network).

The feasibility of large scale separations was piloted employing two
different separation principles: one was the extraction and/or precipi-

tation of the dry matter in a spent sulfite liquor with alcohol, the


Separation by lon Exclusion oe

Several years ago In this laboratory, ploneering research had been
conducted on sugar lignin sulfonate separations using ion exchange resins
(17). A fractionation of SSL components into sugar-rich and lignin sul fon---
ate-rich portions was achieved. In recent years, this technique has been
studfed and improved in several academtc and industrial laboratories (7, 38).....
To evaluate the separation under optimal conditions, we carried out some
studies applying extremely low loadings and very slow flow rates and
established material balances.

Experimental

Freeze dried spent sulfite liquor was dissolved in distilled water,
loaded onto a column filled with Dowex 50W-X2, samples eluted with water,
and fractions collected with an automatic fraction collector. These were
examined using UY absorption at 280 nm and the elution diagram was plotted
(see Figure 23). Each fraction was quantitatively neutralized with sodium
hydroxide, thus determining the acid number. The neutralized sodium spent
sulfite liquor was then concentrated, freeze dried, and the dry matter
content of each sample was determined. The elution diagrams from analyti-
cal gel chromatography using Sephadex G-25 and G-50 were also determined.

Results and Discussions

By using the ion-exchange resin in H-form we expected a better separa-
tion of charged molecules, such as lignin sulfonates, aldonic acids and
sugar sulfonic acids, from non-charged molecules such as mono-, di- and
poly sacchartdes. Due to the low loading and slow flow rate, we observed
complete exchange of ions within the first 5 to 10 inches of the total
column length (7 feet). The remaining separation was exclusively governed

by the principle of fon-exclusion.

68


Figure 22). Two-thirds of the material represented carbohydrate degrada-
tion products. The extracted lignin sulfonate fraction gave a well resolved
elution pattern on gel chromatographic separation using Sephadex G-I5. It
could be demonstrated that the so-extracted LOMOLS fraction consists
mainly of very low molecular weight matertal.

Subsequent extraction with 88% ethanol, carried out in three stages,
resulted in the dissolution of 22% of the organic matter consisting of
about 50% of the lignin sulfonates. The ratio between absorbing (lignin-
!ike) and non-absorbing (carbohydrate-like) material in all three stages
was practically the same. The elution diagrams obtained from analytical
Sephadex separations Indicate that these fractions consist also of very
low molecular weight materials.

In another set of experiments we studied the precipitation of lignin
sulfonates from aqueous solutions by ethanol. We established the material
balance and found that the ratio between carbohydrate degradation products
and lignin sulfonates over the investigated range of ethanol-water mixtures
(up to 20% water) remained constant. Comparative gel-chromatographic LOMOLS
obtained by the two different techniques (extraction and precipitation)
have a very similar composition.

Conclusions

These preliminary studies allow the conclusion that a certain amount
of carbohydrate degradation products (e.g., monosaccharides) can be extracted
with absolute ethanol, or generally, high percentage ethanol (beyond 95%);
however, a satisfactory separation of lignin sulfonates and carbohydrate
reaction products by more extraction with, or precipitation from, ethanol

water mixtures does not seem to be feasible.

67


Although no complete categorical separation of the SSL components
could be achieved, the accomplished separation into three matn fractions
with distinct different composition ts of enormous technical and scientific
interest.

The main fraction comprises about 55% of the total material and con-
tains hfgh and mtddle molecular weight Iftgnin sulfonates, contaminated
with carbohydrate degradation products to an extent of about 10% or less.

The second fraction consists of low and middle molecular wetght lig-
nin sulfonates and carbohydrate degradation products in about equal pro-
portions (1:1). Since the content of reducing groups is moderate, we
assume that the non UV absorbing material consists mainly of aldonic acids.
The fraction amounts to about 15% of the total material.

The remaining 30% contains only traces of lignin sulfonates and com-
prises carbohydrate degradation products with the bulk of mono- and di-
saccharides.

Conclusions

The reported preliminary study demonstrated that a crude separation
of spent sulfite liquor components into three major fractions by ion
exchange exclusion chromatography can be achieved.

From an industrial point of view, all of thé three fractions have a
utilization potential. The last two fractions contain practically all of
the carbohydrate degradation products which may represent a valuable source
for a number of salable products.

Both experiments gave rise to preferential separation of low molecular
weight lignin sulfonates. This is indicated in the graphs of Figure 22

and Figure 23. In the separation with alcohol, 10% of the Itgnin sulfonates

70


TMS
GH2ocochs
H3e o-GH erythro = f.
CHS03CHy
CH 30 mp,
2 = 113"
OCH;
COCHs
A a . . :
20 30 40 50 ppm(r) 60 30 90 10
FIGURE 6.

NMR~SPECTRUM OF A TYPICAL LOMOLS-LIKE DIMER.

26


FIGURE |

Preparation of Spent Sulfite Liquor

Sulfite Pulping

]

Spent Sulfite Liquor
+

Dowex 50W-X8 De-ashing

¥

SO, Stripping

Ether Extraction

t

Ba(0H), Addition; Centrifugation

i

Dowex 50W-X2: lon Exclusion Separations

Purified Lignin Sul fonates

Ca(OH), Neutralization

Sephadex G-25 Fractionation

Low Molecular Weight Lignin Sul fonates

Cellulose Pulps

Ether Solubles

BaS0,, etc.

Carbohydrates
+ Aliphatic - COOH

High and Medium
Mol. Wt. Lig. Sul fonates



A substantial red-shift was evidenced upon alkalination for the lowest
molecular weight lignin sulfonates, which indicates the presence of some
unique functional groups, probably in the side chains of the Co-units of
the sulfonates.

An examination of various fractions by TLC revealed that the sul fon-
ates could be divided into those immobile on TLC plates and those mobi le.

The immobile sulfonic acids formed brown solutions.


vi

t
A280nm

bot

40--

10f-

COMPONENTS IMMOBILE ON TLC-PLATES
0 L i it l
2200 3000 4000 ‘5000
a a a a a 4
A B c Dd FG

1
6000
os &4& & & & & ob
J « LM N O P
EFFLUENT VOLUME, mt

a a
H '

mp

FIGURE 2.

Fractionation of Sulfonates in Spent Sulfite Liquor by Gel Chromatography on
Sephadex G-25 in a Column 10 feet by 2 inches ID with Water as Eluant.


LZ

On

. 2 ieee aoe
Op Pon fe CH

HZ0COCH3
H

busogchy

CH30 468. be
426 OCHS, cho TM, oy acre 331
TMe = Oty Ochs "“e
hao sos Ochs,
item SH, my, oc x gH20ne
of id « Ochs fe
i. cH - 236
chy —_— <*> tt "
331 OCH as My
TM OCs
TM%  @ My bens cus
313 468. (43%)
236.
33
| ase a7 282 300 | 372394 426 |
Mus a ll, A nm A
MASS UNITS
FIGURE 7.

MASS~SPECTRUM AND FRAGMENTATION PATTERN OF A TYPICAL LOMOLS-LIKE DIMER,


These relationships are of particular significance because our
research confirms the concept that the effective size of a lignin sul fon-
ate molecule may be changed substantially by change in the concentration
of electrolyte solution in which the molecule is dissolved.

When the size of the lignin sulfonate molecules falls approximately
within the range of the sizes of the pores in the particular Sephadex
molecular sieve, effective fractionation may occur. In further gel
chromatography fractionations, gymnosperm lignin sulfonates in aqueous
NaC! at 0,001 M and other molarities have been separated into ten sub-
stances or mixtures of substances amounting to about eighteen percent of
the weight of total lignin sulfonates. The ultraviolet spectra of these
was about the same as that of the unfractionated sample. Sedimentation
equilibrium molecular weights of the entities ranged from several hundred
up to about three thousand. The entities appear to be the lower molecular
weight members of the polymer series of lignin sulfonates.

Based on the molecular weights measured for these "peak" substances,
we have speculated that the peaks represent lignin sulfonate mers extending

up to the !0-mer or |2-mer, and possibly the |4-mer (4).


WE |GHT

RECOVERED
$04 fg J

>

hn HIMOLS

Ihe

1200,

leto)

UNI DENT | FLED

800

boo

7* REDUCING

/ SUGARS
20 y,

50 To fo ho ISO So Mo Yo
ELUTION VOLUME

FIGURE 23.
ELUTION DIAGRAM OF A SSL IN ION EXCLUSION CHROMATOGRAPHY.

69


Forss and coworkers found that the UV absorption of the LOMOLS suf-
fered a severe red-shift when the solution was alkalized (7). The UV
spectrum of HIMOLS appeared unaffected by the pH. This Finnish group
also reported findings which demonstrated a significant difference in
solubility in sodium bisulfite solution of pH 5.5 - 6 between Lo and HIMOLS
(6). Fractionation of LS over ion exchange resin (DQWEX 50W-X2) separates
the LOMOLS from the HIMOLS (see Section V). This separation can be visu-
alized also by the color: LOMOLS are yellow, whereas HIMOLS are brown,
Fractionation on Sephadex G-25 yields an elution diagram similar to that
from Dowex. Very significant differences bewteen low and high molecular
weight parts of LS can be observed In the elemental and functional group
composition, as discussed in earlier sections of this report (Section V,
VII, and VIII).

As to the origin of these differences that are in apparent disagree-
ment with the conventional understanding of lignin, (particularly concerning
its homogeneity) (see ref. 27-29), Forss and coworkers believe that fignin
is as heterogeneous as the carbohydrate portion in wood (cf. hemi- and
holo-cellulose). Other theories explain these differences with secondary
condensation of lignin material evoked by acid hydrolysis.

To find a better understanding of the structure, origin, and reactivity
of LOMOLS we undertook two-fold studies:

a. We isolated lignin sulfonates into a series of fractions each of
which consisted of particular mers, and we characterized each mer
class by several methods; and

b. We sulfonated the hydrolysis products of the most representative
dimeric lignin model compound and investigated the reaction

products.
54


TABLES

Characteristics of Ultraviolet Absorption

Chemical Shifts for Acetylated and Esterfied Lignin
Sulfonates in NMR Spectroscopy

Empirical Formulae of ALSEME Derivatives of MWL

Extraction Conditions

viii

Page

25

43
65


spectra of sulfonated lignin models and lignin sulfonate fractions did not
yield satisfactory information mainly due to the tack of solubility in
organic solvents and due to the presence of strong polar groups. By pre-
paring the acetylated methy! sulfonates well resolved spectra could be
obtained. Figure 6 shows a typical example of: the NMR-spectrum of a sul fon-
ated dimer. Following the increasing magnetic field from the left to the
right side of the Figure, the aromatic protons are exhibited first, followed
by the protons of the benzylic carbon atoms which carry acetoxy-groups.
Methine- and methylene-protons of the sidechains of Cy units generally

give rise to signals between the region of the aromatic protons and the
protons of the methoxylgroups, with which they imterfere.

Protons of acetylgroups are much less effected by other signals than
are methoxy-protons. Aromatic acetoxy-protons usually appear at lower
field, well separated from their aliphatic counterparts; however, biphenyl!
and phenyl-alkyl linkages ortho to acetoxy-groups cause a significant
shift of the acetyl-protons to higher field, where they interfere with the
aliphatic acety!-groups.

The average chemical shifts for acetylated and esterified lignin
sulfonates in NMR-spectroscopy are listed in Table 2. This Table provides
the possibility of estimating rather precisely the relative distribution
of protons in LOMOLS-like materials and therefore, allows a nearly com-
plete functional group analysis of the acetylated methyl! sulfonates.

Mass Spectroscopy

Systematical mass spectroscopic studies of lignin related models had

not been reported before we started with our investigations. However,

several papers dealing with the mass spectroscopic fragmentations patterns

24


ci,on

es
CH-OH H®

—s

-H,0

POLYMER

FIGURE Zl.

cH, OH
c=0
!
cH,
—>
-Ar0H

chon

é—on
i}

POLYMER

ACID HYDROLYSIS OF THE VERATRYL-GLYCERYL-/
-ARYLETHER, ITS SULFONATION- AND POLYMERI-

ZATION PRODUCTS.

6


SECTION X
PRELIMINARY STUDIES ON INDUSTRIAL

SCALE SEPARATION OF SSL COMPONENTS

introduction

In the light of the investigations reported in the earlier sections
of this report and prior work in this laboratory, preliminary experiments
were carried out directed toward investigation of feasibility of industrial
scale separation of SSL components.

Two techniques were given preliminary study. One was solvent extrac-
tion of spent sulfite liquor solids by butanol-water and ethanol-water
mixtures. The other technique was a further study of the use of fon-
exchange resins on a column arrangement. LOMOLS are separated from HIMOLS
presumably through some molecular sieve mechanism and from sugars in recog-
nition of the fact that the non-ionic sugars would not be excluded from the
pores In an ion-exchange resin, whereas the lignin sulfonates would be so
excluded.

Earlier work in this laboratory showed that high molecular weights
lignins can be separated from the rest of the substances in SSL, perhaps
technically although not necessarily economically by dialysis (31) and
by the formation of amine salts of lignin sulfonates. Quantitative
separation from sugars was achieved by precipitation or solvent extraction
of the amine salts of lignin sulfonates. This salt formation was reversed
by use of sodium hydroxide or another strong base to give rise to freeing

the amine and formation of the metal salts of the lignin sulfonates which

is perfectly soluble in water.

63


Separation by Alcoho! Extraction

tn thts laboratory, a considerable effort has been made toward
separating lignin sulfonates of various molecular weight (32-36). One
of the approaches has been the fractionation by ethanol-water mixtures (37).
It could be demonstrated that the MW of the dissolved fractions increased
with the water content of the eluant. Preliminary + tests have shown that
SSL solftds can only be extracted with ethano|-water mixtures with water
contents up to 15%. At hi higher water contents, the solids yielded a sticky
unextractable tar.

Experimental

Steam-stripped fermented spent sulfite liquor from gymnosperms was
provided by the Georgia-Pacific Corp., in Bellingham, Washington. The
liquor was neutralized with sodium hydroxide, freeze dried, and then extrac-
ted with ethanol-water mixtures beginning with 95% ethanol and followed by
exhaustive extraction with ethanol containing increasing proportions of
water. The extracts and extracted solids were analyzed by Sephadex gel
chromatography applying controlled ionic strength and the results are shown
in Table 4.

In a second similar experiment spent sulfite |iquor solids (Calcium
base) were extracted with ethanol and ethanol-water mixtures and the weight
of the dissolved solids and the absorbance of solutions of the same was
determined. A material balance by weight and absorption at 280 nm was
established.

Results and Discussion

Treatment with 95% ethanol resulted in the extraction of about 2% of

the organic matter which contained only 25% UV absorbing matertal (see

64


I rs) Se 6
CHSO3Me CHSO3Me
——
OCH
OCH , 3
on OCOCH3
R 66 R ! e,e
CHSO3Ag CHSOSH
—oO
OCH OCHs
OCOCH3 OCOCH3
“|
CHSO3CH3
OCH3,
OCOCH3
FIGURE 4.

REACTION SCHEME FOR THE TRANSFORMATION PROCEDURE.


3.

10.

MW.

12,

SECTION X11}
REFERENCES

Schubert, S. W., Audrus, M. G., Ludwig, C., Glennte, D., and

McCarthy, J.L., "Crystalline Low Molecular Weight Lignin-Type
Sulphonates. 11. Synthesis and Establishment of Structure."

TAPPI, 50, 186-193 (1967).

Glennie, D. W., "Chemical Structure of Lignin Sulphonates. III.

Some Reactions of Monomeric Lignin Sulphonates.'' TAPP! 49, 237-
242 (1966) . ——

Gupta, P. R., and McCarthy, J. L., "Lignin XIV. Gel Chromato-

graphy and the Distribution in Molecular Size of Lignin Sulphonates
rH on Electrolyte Concentrations," Macromolecules, 1, 236-244
1968). ~

Gupta, P. R., and J. L. McCarthy, "Lignin XV. Preliminary Char-
acterization of Several Low Molecular Weight Ligninsulfonate
Mers,"' Macromolecules, 1, 495-498 (1968).

Forss, K., ''The Composition of a Spent Spruce Sulfite Liquor,"
Dissertation, Abo Akademi, 1961.

Forss, K. and Fremer, K. E., "The Dissolution of Wood Components
Under Different Conditions of Sulfite Pulping," TAPPI, 47(8),
485-493 (1964).

Jenson, W., Fremer, K. E. and Forss, K., '"'The Separation of the
Components in Spent Sulfite Liquor," TAPPI, 45(2), 122-127 (1962).

Ludwig, C. H., Nist, B. J., and McCarthy, J. L., "Lignin XII. The
High Resolution Nuclear Magnetic Resonance Spectroscopy of Protass

in euyrounds Related to Lignin,'' J. Amer. Chem. Soc., 86, 1186-1196
(1964).

Ludwig, C. H., Nist, B. J., and McCarthy, J. L., "Lignin XIV. The
High Resolution Nuclear Magnetic Resonance Spectroscopy of Protass
In Acetylated Lignins,"' J. Amer. Chem. Soc., 86, 1196-1202 (1964).

Lenz, B. L., “Application of Nuclear Magnetic Resonance Spectro-
scopy to Characterization of Lignin," TAPPI, 51, 511-519 (1968).

Kovacik, V., ''Massenspectrometric Einiger Modellsubstanzer des
Lignins, 1,'' Chem. Ber., 102, 1513-1522 (1969).

Kovacik, V., ''Massenspectrometric Einiger Modellsubstanzer des
Lignins, 11," Chem. Ber., 102, 3623-363! (1969).

77


Section

vi

Vit

vill

Xl
xt
XIII
XIV
XV

CONTENTS

Conclusions
Recommendations
Introduction
Objectives and Plans

Preparation, Fractionation and Preliminary Characteriza-
tion of Gymnosperm Lignin Sul fonates

Preparation of Certain LOMOLS-!ike Monomers and Dimers and
Characterization by NMR and Mass Spectroscopy

Preparation of Acetyl Lignin Sulfonate Methy! Ester (ALSME)
and their Spectroscopic Characterizations

Structural Studies of Acety! Lignin Sulfonate Methyl Esters
Derived from Milled Wood Lignin

Structure and Reactivity of LOMOLS

Preliminary Studies on Industrial Scale Separation of SSL
Components

Concluding Comments
Acknowledgements
References

Publications and Patents

Glossary

29

39

53
63

73
75
77
81
83


second was the fractionation of the material by ion exclusion in a
column arrangement. Both methods seem to allow the separation of
basieally three different classes of substances, namely, high molecular
weight LS, low molecular weight LS, and carbohydrate degradation

products.


SECTION IV

OBJECTIVES AND PLANS

Our general objective was to find new information concerning lignin

sulfonates, and especially those of low molecular weight, which would
provide a better foundation for development of profitable utilization
or the innocuous and economic disposal of these materials, and of the

spent sulfite liquors in which they are contained.

The specific aims of the research program were:

(a) To secure further knowledge of how to purify low molecular weight
lignin sulfonates by separating them from other classes of materials, -

such as higher molecular weight lignin sulfonates, sugars and carbohydrates,
inroganic substances, etc,

(b) To learn how to separate purified lignin sulfonates Into fractions

of differing molecular weights with certain factions hopefully to consist
mainly of particular lignin sulfonate mers, such as monomers, dimers,
trimers, and higher mers. It was anticipated that some crystalline monomers
might be obtained but mainly it was expected that a mixture of several
different mers would be found in each fraction;

(c) To characterize each lignin sulfonate fraction by a number of spectro-
scopic and other available procedures in order to secure additional
knowledge of the chemical structure and reactions of lignin sulfonates.

(d) To prepare and characterize derivatives of low molecular weight

lignin sulfonates to augment the information gained by characterization

of the lignin sulfonates themselves;

(e) To try to find new approaches to the economic utilization of lignin

sulfonates.


19

20

21

22

23

THE "REGENERATION PATHWAY'' IN TERMS OF PEAKS, AS
VISUALIZED FROM THE SEPHADEX LH-20 FRACTIONATIONS

ELUTION DIAGRAM OF BROMINATED ES ‘ALSME DERIVATIVES~ ><
AND THE REFRACTIONATION PATTERNS OF SOME MER CLASSES

ACID HYDROLYSIS OF THE VERATRYL-GLYCERYL-B-ARYLETHER,
ITS SULFONATION AND POLYMERIZATION PRODUCTS

DISSOLUTION OF SSL SOLIDS AS A FUNCTION OF THE WATER
CONTENT OF ETHANOL ta .

ELUTION DIAGRAM OF A SSL IN ION EXCLUSION CHROMA-
TOGRAPHY

vii

Page
59

- 60

6l

66

69


26. Gierer, J., "The Reactions of Lignin During Pulping. A Des-
cription and Comparison of Conventional Pulping Processes,"

Svensk Papperstidning, 73, 571-596 (1970).

27. Freudenberg, K., "Uber einen Foromelvarschlag fur das
Fichtenlignin," Holzf., 22, 65-66 (1968).

28. Forss, K., "Das Fichtenlignin," Holzf., 22, 66-68 (1968)
29. Freudenberg, K., "Antwort," Holzf., 22, 68-69 (L968).

30. Adler, E., "Guaicylglyceral and its B-Guaicyl Ethyl." Acta
Chem. Scand., 9, 341-342 (1955).

31. (i) Lundquist, K., "On the Separation of Lignin Degradation
Products,"' Acta Chem. Scand., 18, 1316 (1968).

(if) Lundquist, K., "Isolation of 3-Hydroxy-1-(4-hydroxy-3-
methoxy pheny!)-2-propanone from Lignin,'' Acta Chem. Scand.,
16, 2303-2304 (1962). ee

32. Peniston, Q. P. and McCarthy, J. L., "Lignin |. Purification
of Lignin Sulphonic Acids by Continuous Dialysis,'' J. Amer.
Chem. Soc., 70, 1324-1328 (1948).

33. Moscanin, J., Felicetta, V. P., Maller, W. and McCarthy, J. L.,
"Lignin Vi. Molecular Weights of Lignin Sulphonates by Light
Scatterings,"' J. Amer. Soc., 77, 3470-3474 (1955).

34. Felicetta, V. P., Ahola, A., and McCarthy, J. L., "Lignin VII.

Distribution in Molecular Weight of Certain Lignin Sulphonates,''
J. Amer. Chem. Soc., 78, 1899-1904 (1956).

35. Mokihara, E., Tuttle, M. J., Felicetta, V. P., and McCarthy, J. L.,
"Lignin VIII. Molecular Weights of Lignin Sulphonates during
Delignification by Bisulfite-Sulfurans Acid Solutions,'"' J. Amer.

Chem. Soc., 79, 4495-4499 (1957).

36. Felicetta, V. P., and McCarthy, J. L., "Lignin IX. Molecular
Weights of Lignin Sulfonates as Influenced ty Certain Acidic
Conditions," J. Amer. Chem. Soc., 79, 4499-4502 (1957).

37. Moscanin, J., Halson, H., Felicetta, V. P., and McCarthy, J. L.,
“Lignin XI. Estimation of Polymolecularity in Lignin Sulfonate
Polymers from Diffusion Measurements,'' J. Amer, Chem. Soc., 81,

2054-2056 (1959).

38. Markham, A. E., Feniston, Q. P. and McCarthy, J. L., ''Lignin III.
Fractional Precipitation of Barium Lignin Sulfonates from Water
vs. Ethanol,'' J. Amer. Chem. Soc., 71, 3599- 3607 (1949).

39. Benko, J., U. S. Patent No. 3,509,121, April 28, 1970, Dryden
Chemicals Ltd., Oakville, Ontario, Canada.

79


SELECTED WATER 3. Accession No.
RESOURCES ABSTRACTS

INPUT TRANSACTION FORM
4. Title

Studies of Low Molecular Weight Lignin Sulfénates

7. author) B. F. Hrutfiord, L. N. Johanson, J. L. McCarthy,

K. Forss, W. Glasser, J. Gratzl, J. Collins 10. Project No.
University of Washington AL. Contract/Grant No.
\ 12040 DEH
. ape

‘of

15. Supplementary Notes

Environmental Protection Agency Report No. EPA-660/2-74-069, June 1974

16. Abstract Low molecular weight lignin sulfonates have been separated in purified form and
characterized by physicochemical and chemical methods. Their structure and reactions
have been evaluated.

Lignin sulfonates from the spent sulfite liquor of a mild acid bisulfite cook of
Western Hemlock (Tsuga heterophylla) were purified and fractionated in Sephadex G-25
column chromatography. Samples were analyzed using acetylation of hydroxyl-groups and
esterification of sulfonate-groups which aided the elimination of the polydisperse
nature of the material under investigation.

Complete elemental and functional group compositions were established for lignin
sulfonates from a spent sulfite liquor and compared to those from milled wood lignin
preparation. This allowed an estimate of the degree of sulfonation, condensation and
demethylation as well.-

Extended separation studies indicated the low molecular weight lignin sulfonates
be the reaction product of a difunctional vinyl-type polymerization, thus accounting
for the widely different properties as compared to their higher molecular weight
counterparts. The feasibility of large scale separations was determined using (1)
the extraction and precipitation of the dry matter in a spent sulfite liquor with
alcohol, and (2) the fractionation of the material by ion exclusion in a column
arrangement.

17a. Descriptors
Sulfonates, lignin, pulp wastes, chemical: analysis, by-products, molecular structure,
organic wastes, separation techniques, sulfite liquors, waste identification, Pacific
Northwest U.S. 7

17, Identifiers

Lignin sulfonate separation, chemical composition, spent sulfite liquore

17c. COWRR Field & Group

18, Availability “{] Send To:

WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
{| WASHINGTON, D.c. 20240

Abaactor_H. Kirk Willard Tostitution EPA - Pacific NW Environmental Res. Lab.

WRSIC 102 (REV. JUNE 1971! “@PO 488-935



TABLE |

CHARACTERISTICS OF ULTRAVIOLET ABSORPTION
SPECTRA OF FRACTIONS A - P_IN FIGURE 2

Fraction Neutral Solution Alkaline Solution
Maximum Minimum Maximum
A 281 261 282
B 281 261 282
Cc 281 260 282
D 280 259 286
E 280 258 286
F 280 258 286
G 280 258 288
H 280 258 290
| 280 257 291
J 280 256 293
K 280 256 293
L 281 261 298
M 282 262 299
N 282 262 299
0 282 262 299
P 282 260 298

These studies showed that the fractions up to an effluent volume of
3,200 ml contained only solutes immobile on the TLC-plates. The fractions
in the range 3,200 - 4,100 ml contained immobile as wel! as mobile compon-
ents. In the fraction range 4,100 - 7,600 ml the fractions contained only
mobile components.

Each fraction containing mobile components formed two or three spots
on the TLC-plates. The location of these spots is indicated in Figure 2.
A comparison with Figure 3 shows that the immobile solutes are to be found

almost completely in fraction group | that contains only small amounts of

16


EPA-660/2-74-069
June 1974

Environmental Protection Technology Series

eee sem 4 .
|
|
|



SECTION VIII
STRUCTURAL STUDIES OF ACETYL LIGNIN SULFONATE
METHYL ESTERS (ALSME) DERIVED FROM MILLED WOOD LIGNIN
Introduction

The separation of lignin sulfonates from spent liquors of sulfite
pulping processes has been studied extensively In this laboratory during
previous years (3, 4, 17). The major obstacle has been repeatedly shown
to be a complete separation of aromatic substances derived from sul fona~
tion and dissolution of lignin tn wood, from the sugars which are formed
at the same time by acid hydrolysis of carbohydrates (17). Some sort of
separation based on the ionic strength of the compounds to be separated
(ion exclusion separation) seems to provide the best method so far (3, 17,
18); however, neither the lignin nor the carbohydrate part can be obtained
absolutely pure and free of contaminations by the ton exclusion method.
Ten-20% contaminations of the respective counterpart seems to be a rea-
sonable goal to be achieved by fon exclusion chromatography. Complete
separation of aromatic- and sugar=components and complete recovery of both
parts from spent sulfite liquor (SSL), both of which are requirements for
a scientific investigation of structural phenomena, had been out of reach
with the methods available so far.

To get a better founded and more complete approach to our research
objectives, it appeared necessary to study the sulfonation reaction (as
used in the acid sulfite pulping process of wood) with a variety of models
for lignin in wood (See Section VI).

-We started thts approach with the preparation of various LOMOLS~like
monomers and dimers. Thetr sulfonation and transformation to acetylated

39


Preparation of the ALSME fractions succeeded. These materials gave rise
to NMR spectra which yielded further information concerning the structure
of the lignin sulfonate molecules of differing molecular weights.
Especially significant ts the finding of an orderly change in phenolic

hydroxy! content as a function of the molecular weight of lignin sulfonates,

Generally, the low molecular weight lignin sulfonates appear to arise
perhaps mainly as hydrolysis products of high molecular weight lignin
sulfonates, although some condensation or repolymerization of the low

molecular weight entities seems to occur simultaneously.


SECTION !

CONCLUS | ONS

Low molecular weight lignin sulfonates can be separated in purified form
from most other materials in spent sulfite liquors of the acid bisulfite
pulping process by use of jon exclusion processes functioning in packed
column equipment. Selective extraction or precipitation with alcohol of
lignin sulfonates in aqueous solution does not yield particularly useful

results.

Separation of purified lignin sulfonates into fractions of differing
average molecular weights can be conducted effectively on a laboratory
basis in aqueous solution by use of gel permeation with molecular sieve

materials such as Sephadex 25 contained in a column.

The separated lignin sulfonates. from two separate sources (one from a
commercial spent sulfite l!iquor, and the other from a sulfonated milled
wood lignin) showed gel chromatography elution patterns, ultra violet
absorptions and certain other characteristics which were quite similar

to each other in view of the differences in the two preparation procedures.

It was concluded that further useful characterization of the lignin
sulfonate fraction might be accomplished by using a derivative soluble In
organic solvents and therefore a procedure was developed for preparation

of acetylated lignin sulfonate methy! esters (ALSME).

To provide experience toward synthesizing the ALSME preparations, acety-
lated methyl sulfonate ester derivatives were prepared for some eighteen
different lignin sulfonate-like monomers and dimers. These were
characterized by chemical analyses, NMR spectroscopy and mass spectroscopy.


SECTION IX

STRUCTURE AND REACTIVITY OF LOMOLS

Introduction

The structure of LS has been elucidated so far nearly exclusively
on the basis of either chemical reactions of lignin models, or physico-
chemical characterizations of LS by ge! permeation chromatography (see
Section ¥, Vf}. [+t has been our objective in this research enterprise
to separate these isolated LS into a series of fractions each of which
consist mainly of particular mers, such as monomers, dimers, trimers, and
then mixtures of higher mers, and to characterize each mer class by several
methods (see Section [Y).

We approached these objectives by purifying and fractionating LS
from a SSL over Sephadex G-25 (see Section V).. Characterizations by UV
and {Rpreceded the transformation of the various LS fractions to ALSME
derivatives (Section Vit). The analytical basis for the characterization
of such ALSME derivatives had been created using LOMOLS-like monomers and
dimers as models (Section VI). The use of Milled Wood Lignin as an “ad-
vanced model" for wood allowed us to make concise statements about the
elemental composition of lignin before and after sulfonation, and the fate
of most functional groups during this reaction (Section VIII).

From the literature and from our earlier studies, we knew that LOMOLS
appeared different from the rest of the LS by a number of characteristics,
as reflected in various chemical and physicochemical methods. The most

important ones of them shal! be listed in the following.

53


Characterization by IR Spectroscopy

The IR spectra of ALSME derivatives are all very similar and without
characteristic features. The sulfonate group exhibits a strong band
around I and 12 , which is ‘rather untypical. The acety! groups raise
strong ester bands fn the nefghborhood of 5.8 , thus covering eventual ly
present carbony! groups. These carbonyl groups, however, can be detected
in the IR spectra of the untransformed fractions. It could be shown that
the carbony! content of the LOMOLS is usually very low. Some fractions,
however, in the very low molecular weight range exhibit a distinct con-
centration of CO-bands (See Figure.11). No attempts were made to investi-
gate the nature of the carbony| groups, which might also be due to incomplete
separation from carbohydrate degradation products.

Conclusions

The polyelectrolytic and polydisperse nature of the LOMOLS could be
eliminated by a relatively simple transformation procedure that covered
the highly polar groups. We assume that this transformation did not induce
any particular changes in the original structures of the LOMOLS. The so-
obtained ALSME derivatives were found to be soluble in organic solvents,
which aided considerably their analysts.

The most valuable analytical method proved to be NMR spectroscopy. It
could be demonstrated that the NMR spectra of low molecular ALSME deriva-
tives provide information on structures which could not be obtained from
other analytical and spectroscopic methods so far. Indications on typical
side chain structures and types of condensations can be as well derived
as an almost complete functional group analysis. It could be demonstrated

that the content of aliphatic and phenolic hydroxy! groups can be estimated

35


SECTION XIV

PUBLICATIONS AND PATENTS

The following publications have been produced or are anticipated to be

produced as a result of this project:

SPENT SULFITE
LIQUOR XIV.

LIGNIN XVI.

LIGNIN XVII.

Preparation, Fractionation, and Preliminary
Characterization of Cymnosperm Lignin
Sulfonates from Spent Sulfite Liquor and
Milled Wood Lignin. Forss, K., Collins, J.J.,
Glasser, W.G., Gratz!, J.S., and McCarthy,
JeL., Tappi, 55, 1329-1333 (1972).

Synthesis, Nuclear Magnetic Resonance, and
Mass Spectroscopy of Several Monomeric and
Dimeric Lignin-like Sulfonates. Glasser,
W.G., Gratzl, J.S., Collins, J.J., Forss, K.,
and McCarthy, J.L., Macromolecules, 6,

(14 €1973)

Preparation and Characterization of Acetyl
Lignin Sulfonate Methy! Esters. Glasser,
W.G., Gratzl, J.S., Collins, J.J., Forss, K.,
and McCarthy, J.L. To be submitted for
publication in Macromolecules.

It appears that no patentable developments have arisen from the

present study.


R
TYPE 1

——_ HSO3CHs R= ALKYL
‘OCH. CHa
byetner 3 I Rew
Ae basof
cHasos”
CHZOR, i
" OCH HOR AH
CH OR
SULFONATION
e
frases crs
she R
‘OCH
(CH30 Te 3 duo R=H (SYRINGYL)
2 R= CH (VERATRYL)
RH or BENZOYL
Roz H or CH i i ii
TYPE 2 TYPE 3 TYPE 4
CH:
Le
2
by
le
pital Gae
GAA Re one ie
CHSO;CHs CH30 S02 CH—CH
ae) r
ocu, OCHS Hs
ofginer Oke

FIGURE 5.

THE FOUR TYPES OF LOMOLS-LIKE MONOMERS AND DIMERS
SYNTHESIZED.

23


lignin sulfonate methyl ester (ALSME) derivatives taught us first the
principal sulfonation pathway of compounds with different functional
groups and active sites, and second, the potential use of highpowered
analytical tools such as NMR= and mass-spectroscopy.

The application of these synthetic and analytical approaches to
vartous LOMOLS-fractions secured from SSL by Sephadex G-25 separations
has been discussed in Section Vil in detail.

Milled wood lignin (MWL) (19-2!) is the genuine plant material,
Isolated from wood by a special mild procedure (ball milling and dioxane
extraction). It is free of carbohydrates and extractives, yet widely
consideréd to be unchanged lignin. It is a "model" by definition, since
it comprises only about 25-50% of the total lignin-part In wood, and
since It has another physical form and environment than In wood. The
lack of Incrustation In a cell wall or between cel! walls (middle
lamella) affects the melting and softening behavior of the material
and lets questions of accessibility appear irrelevant. The different
solubility of the isolated lignin allows most reactions to proceed as
homdgeneous reactions (22),

The advantage of working with MWL as a model for lignin (the DP of
MWL is reported to be around 60, molecular weight about 11,000 ts based
on the fact that MWL is a homogeneous material, free of carbohydrates and
extractives. An empirical formula, based on the assumption of Co- monomeric
units, can be designed for the material and a balance of certain functional
groups can be established. That means that the starting naterial of the
reaction (e.g., sulfonation) can be well defined. Changes that occur

during the reaction can be wel! detected and controlled.

40


As shown in Figure 4, sodium sulfonates were acetylated by pyridine/
acetanhydride. The aqueous solution of the free acids prepared by cation
exchange was neutralized by silver oxide. Reaction with methy! iodide
yielded the corresponding acetylated methy! sulfonates.

The 18 model compounds that were synthesized in the course of this
study are represented by four types of monomeric and dimeric lignin build-
ing units, as shown in Figure 5.

Type 2 represents monomers of the guaiacy!l- and veratryl- series with
saturated and unsaturated sidechains. It includes all three possible reac-
tion products of the coniferylalcohol-type structure. Among them is one
a - and one y- sulfonic acid, each with a double bond, and one a-y -
disulfonic acid. All of the three types of sulfonic acids have been iso-
lated earlier from sulfite liquors (1, 2),

The second type of linkage is expected to represent the major portion
of linkages present in lignin. The most important one of them is the con-
figuration which carries an aryl-ether structure in 8-position and a
hydroxy! group in Y-position. Other substituents to consider at the B-
carbon atom are a hydroxylgroup or a carbon-to-carbon !inked second Cy
unit as it is formed from the coumaran structures in lignin in a neutral
sulfite cook. A compound with the latter configuration was supplied to us
by Dr. Josef Gierer from Sweden. The fourth type of linkage represents the
dipheny!structure.

NMR-Spectroscopy

Extensive studies performed earlier in this laboratory (8, 9)

revealed that NMR-spectroscopy represents a valuable method to study the

overall structure of different lignin preparations, However, the NMR

22


Effluent Volume

Cc

refract |

refractd

A.s0

aouequosqy

aouequosqy

Effluent Volume

FIGURE 20.

Elution Diagram of Brominated ES ALSME Derivatives and the

Refractionation Patterns of Some Mer Classes.

60


210

W

12

13
14

15
16
17
18

FIGURES

PREPARATION OF SPENT SULFITE LIQUOR

FRACTIONATION OF SULFONATES IN SPENT SULFITE LIQUOR
BY GEL CHROMATOGRAPHY ON SEPHADEX G-25

SHIFT OF THE WAVELENGTH OF THE ABSORPTION MAXIMUM
NEAR 280 nm ON ALKALIZING FRACTIONS OF THE SPENT
SULFITE LIQUOR .

REACTION SCHEME FOR THE TRANSFORMATION PROCEDURE

THE FOUR TYPES OF LOMOLS-LIKE MONOMERS AND DIMERS
SYNTHESIZED

NMR-SPECTRUM OF A TYPICAL LOMOLS-LIKE DIMER

MASS~SPECTRUM AND FRAGMENTATION PATTERN OF A TYPICAL
LOMOLS-LIKE DIMER

NMR SPECTRA OF ALSME DERIVATIVES, PREPARED FROM VAR-
IOUS FRACTIONS FROM SEPHADEX G-25 SEPARATIONS

SEPHADEX LH-20 SEPARATIONS OF SOME ALSME FRACTIONS
1N METHANOL

CORRELATION BETWEEN PHENOLIC HYDROXYL GROUPS AND ARO-
MATIC PROTONS IN FRACTIONS FROM SEPHADEX SEPARATIONS

THE CARBONYL REGION IN IR-SPECTRA OF FRACTIONS FROM
SEPHADEX SEPARATIONS

HYPOTHETICAL DIMER FORMATION MECHANISM
ELUTION DIAGRAMS OF CALCIUM LIGNIN SULFONATES

ELEMENTAL AND FUNCTIONAL GROUP COMPOSITION OF MWL-
ALSME

SCHEME 3
SCHEME 6
OXYGEN FUNCTION IN ALSME DERIVATIVES OF MWL

ELUTION DIAGRAM OF ES ALSME DERIVATIVES AND THE
REFRACTIONATION PATTERNS OF SOME MER CLASSES

vi

Page

2i
23

26
27

30

32

34

36

38
42
45

46
48
49
56


continuous UV recording apparatus, then neutralized with calcium hydroxide
and concentrated by evaporation. This CaSSL was subjected to Sephadex G-25
gel chromatography and the absorbance at 280 nm of the resultant elution
liquid was recorded and is shown in Figure 2.

The relatively smal! size of the peak in the effluent volume range
2,200 - 2,600 ml in Figure 2 shows that the spent sulfite liquor contained
only a small proportion of lignosulfonates excluded by Sephadex G-25, i.e.
lignosulfonates with molecular weights larger than 10,000 (7). This is
in all probability due to incomplete delignification during the cook that
was performed in order to produce a high proportion of low-molecular-weight
sulfonates.

The eluted liquid was divided into a series of sixteen fractions
(A-P} and in each case, an overlapping peak was defined by thin layer
‘chromatography using isopropanol-water developer. The mid-section of
corresponding peaks for a series of runs were combined and freeze dried.
The resultant material was subjected to analysis by ultraviolet and infra
red spectroscopy.

Characterization by UV-Spectroscopy

Ultraviolet absorption spectra of a selection of fractions marked
A - P in Figure 2 were recorded on a Cary registering spectrophotometer
in neutral and alkaline solution. The overall shape of the spectra
obtained is well known to lignin chemists and therefore only the wave-
lengths of the absorption maxima and minima are shown in Table l.

As expected, all fractions A - P show in neutral solution an absorp-
tion maximum near 280 nm. A closer examination reveals, however, that

fractions A - C exhibit the maximum at 281 nm whereas the maximum in

13


SECTION XV

GLOSSARY
ALSME - Acetylated lignin sulfonate methyl] ester.
Ca/Na-SSL - Calcium or sodium base spent sulfite liquor.

DP - Degree of polymerization.

m

IS - Bther insoluble.

fx}

PC - Gel permeation chromatography.

=x

IMOLS - High molecular weight lignin sulfonates.

LOMOLS - Low Molecular Weight Lignin Sulfonates.

LS - Lignin Sulfonates.

MW - Molecular Weight

NMR = Nuclear magnetic resonance.
SSL - Spent sulfite liquor.

TLC - Thin layer chromatography

UV - Ultra violet spectroscopy.

83


(see Section VI and VII). Side chain configurations as well as ring
substituents can be estimated or determined with high accuracy.

The application of NMR spectroscopy to the 12 fractions of the MWL
fractionation revealed some more specific features of the structure of LS.
Fractions 2 and 3 proved to contain some sulfonation products of coniferyl-
alcohol (Scheme 5, Figure 16).

These compounds, however, were very probably not present as monomers
but rather attached to oligomertc fragments as indicated by thin layer and
Sephadex LH-20 chromatography. The C-6 atom of the aromatic ring is the
position of intermolecular linkage. Since both vinyl-compounds have only
three oxygen functions per Cg, not all oxygen deviation shown in Figure 17
can be due to condensation; some of it must be due to these unique side
chain configurations.

The NMR spectra of fractions 8 upward show signs of increasing inten-
sity of carbon-carbon condensation in position 5 of the aromatic ring.
Condensation has been indicated also by the oxygen deviation of these
fractions, shown in Figure !7.

Conclusions

Sephadex chromatography, elemental analysis and NMR spectroscopy
helped to elucidate some of the structure of LS.

It was shown that phenolic hydroxyl-groups are formed through demeth-
ylation of aromatic methoxy!-groups (Scheme 2, Figure 15). In the lowest
molecular fraction of the lignin sulfonates from MWL, about every second
Cy unit was demethylated. The para positions of these newly formed
phenolic hydroxylgroups (position 6 of the Cg unit) are the most activated

nucleophilic sites of the entire lignin (Scheme 6, Figure 16).

50


13.

16.

17.

20.

21.

22.

23.

24,

25.

King, E. G., Brauns, F., and Hibbert, H., "Studies on Lignin and
Related Compounds XVIII. Lignin Sulpharic Acid - A Preliminary
Investigation on Its Isolation and Structure," Can. J. Research,
13 B, 88-92 (1935).

Parrish, J. R., "Monomeric Lignin Sulphonic Acids. Part II,"
J. Chem. Soc. (C), 1145-1150 (1967).

Gellerstedt, G., and Gierer, J., ''The Reactions of Lignin During
Neutral Sulphite Cooking. Part 1. The Behavior of B-Arylether
Structures," Acta. Chem. Scand., 22, 2510-2518 (1968).

Gellerstedt, G., and Gierer, J., ''The Reactions of Lignin During
Neutral Sulphite Cooking. Part 11. The Behavior of Phenyleoumaran
Structures," [bid., 22, 2029-2031(1968).

Felicetta, V. F., Lung, M. and McCarthy, J. L., ''Spent Sulphite
Liquor VII. Sugar-Lignin Sulphonate Separations Using lon Ex-
change Resins," TAPP], 42, 496-501 (1959).

Benko, J., "Separation of Solutions of Spent Sulphite or Kraft
Liquors and Bark Extracts,'' U. S. Patent No. 3,509,121, April 28,
1970.

Bjorkman, A., "Studies on Finely Divided Wood, Part 1|.'' Svensk
Papperstidning, 59, 477-485 (1956). ~~

Bjorkman, A., and Person, B., ''Studies on Finely Divided Wood,
Part 11." Ibid, 60, 158-169 (1957).

Bjorkman, A., "Studies on Finely Divided Wood. Part lil. (i) Extrac-

tion of Lignin Carbohydrate Complexes with Neutral Solvent,"
Ibid, 60, 243-251, 285-292, 329-335 (1957).

(ii) Studfeszon Finely Divided Wood. Part IV. Some Reactions
of the Lignin Extracted by Neutral Solvents from Picea abies.
lus.

(iif) Studies on Finely Divided Wood, Part V. The Effect of
Milling.

Bjorkman, A., Grenoble - Symposium,

Lundquist, K. and Ericsson, L., "Acid Degradation of Lignin VI.
Formation of Methanol," Acta Chem, Scand., 25 756-758 (1971).

Forss, K. and Fremer, K. E., '' The Repeating Unit in Spruce
Lignin," Paperi ja Puu, 47, 443-454 (1965).

Forss, K., Fremer, K. E., and Stenlund, 8., '' Spruce Lignin and

Its Reactions in Sulfite Cooking - 11. The Reactions In Sulfite
Cooking," Paperi ja Puu, 48, 669-676 (1966).

78


naming all peaks according to their retention volume, certain “regenera-
tion pathways" could be established (see Figure 19).

The Tsolation of compound c seemed to be most likely for several
reasons: The NMR-spectrum tndicated the predominant presence of a compound
with a typical vinyl stde chain. This compound had earlier been found to
comprise the major portion of the LOMOLS. TLC demonstrated that this com-
pound was present as monomer (comparison with an authentic sample with this
structure). However, the refractiénation of peak c yielded the desfred
material only in minor amounts, and the fractionation pattern exhibited
resembles strikingly much the original elution pattern of the ES ALSME
derivatives.

These results allowed the conclusion that some compound, possibly the
one with thef-vinyl structure, rearranges and/or isomerizes extremely
readily to nearly everything we call LOMOLS. Since the isomerization
behavior of compound | had been studied before, a reaction pathway fnvoly-
ing a double bond was suspected; therefore, the second half of the ES ALSME
was brominated prior to its fractionation,

After bromination, the only obviously altered peak appears to be d.

It is interesting to note that this peak was found to be the direct pre-
cursor of the polymerization (vinyl-plym.), as stated from the previous
experiment (see Figure 19).

Refractionation of the mer class representing compound d, resulted
indeed in the isolation of a single, stabilized peak d. The refractionation
of the other mer classes showed again the known instability (see Figure 21).
No satisfactory explanation was found for the observation that the vinyl

compound | was again present in the brominated ES ALSME. There ts no reason

58


FRACTION 9- (3 L

FRACTION 6

=

FRACTION |

20 3.0 40 5.0 60 20 80 90
PPM (7)

FIGURE 8.

NMR SPECTRA OF ALSME DERIVATIVES, PREPARED FROM VARIOUS FRACTIONS FROM SEPHADEX
G-25 SEPARATIONS. 30


were separated and proved to be of low molecular weight and relatively

low content of sugar components. tn the untque ion exclusion separation

similar results were obtained as shown jn Figure 23.

Of special interest in most cases are the results obtatned on separa-
tion of thé resultant low molecular weight Itgnin sulfonate fraction when

subjected to gel chromatography using Sephadex G-25 or Sephadex G-50.

The results from the fon exclusion experiment demonstrated that three

different types of fractions could be obtained:

Fraction | represented about 55% of the total material containing
high and medium low molecular weight LS contaminated with carbohydrate
degradation products up to 15%.

Fraction 11 consisted of low to middle molecular weight LS and about
50% carbohydrate degradation products, most of which were non-reducing and
of unknown structure (possibly aldonic acids).

Fractton IIl contained mainly sugars.

7I


The transformation to ALSME derivatives resulted also in a purifica-
tion of the original lignin sulfonate fractions obtained from the previ-~-
ously described Sephadex fractionation (See Section V), which were contam-
inated with carbohydrate degradation products to a certain degree.

NMR Spectroscopy: The spectra were recorded using the same conditions
as described for the LOMOLS-like monomers and dimers (See Section VI).

The entire frequency range of each spectrum was divided Into several areas
that were classified on the basis of the model studies. As shown in
Figure 8, the LOMOLS exhibited well resolved spectra in a wide range of
the investigated fractions.

Chromatography: The separation of ALSME fractions using the cross-
linked Sephadex ge! LH-20 and methanol as eluant resulted in the fraction-
ation of low molecular weight mixtures into simple mixtures and into a
single, crystalline compound (m. p. 200-201°C).

Analytical thin-layer chromatographic studies and fractionations by
Sephadex LH-20 ge! chromatography in analytical scale indicate that the
fractions are composed of more or less complex mixtures of low molecular
weight compounds ranging from monomers to dimers and trimers and some
higher mers (See Figure 9).

Characterization by NMR Spectroscopy

In general, the spectra of all thirteen fractions studied allowed a
good correlation between the protons of various functional groups. Special
emphasts was placed upon the content of aliphatic and phenolic hydroxy!
groups. The correlation between these two hydroxy! fractions led finally
to a classification of the fractions in four different groups. Constant

ratios--allphatic to phenolic hydroxy!s--ranging from 0.5 (fractions | to 7

3I


Haty

Hap
H Ca-0 R
H 8
och3 ———>
OCH3
Scheme 1
l.
6 2 HO HSO3-
3 —————“~
OCH3 ~CH20H?
o—
Scheme 2
| |
CHOH cH
—_—
roms OCH
~ On
Scheme 3

FIGURE 15

46

Hac-
Hat
Hco + HO
OCH3
o—
OH
o—

OCH



SECTION VI

PREPARATION OF CERTAIN LOMOLS=LIKE MONOMERS AND
DIMERS AND CHARACTERIZATION BY NMR AND MASS SPECTROGRAPHY

Introduction

Preliminary Investigatton of the fractions secured from the Sephadex
separations, as described in Section V of this report, indicated the need
for application of the more refined analytical methods that are nowadays
commonly used throughout organic chemistry. Whereas, NMR- and Mass-
spectroscopy had been successfully applied to study lignin and lignin-
like derivatives (8 - 12), these common methods could not hitherto be
used for lignosulfonates.

However, to apply these procedures, it was necessary to secure infor-
mation concerning the spectra or patterns associated with particular
groupings expected to be present in the LOMOL type materials. It there-
upon became necessary to assemble and/or synthesize certain LOMOLS like
monomers and dimers in order to permit the spectroscopy to be carried out
in consideration of the special chemical groupings of interest.

In contrast to other organic natural compounds, the analytical basis
for the characterization of lignin sulfonates remained widely restricted
because of the polyelectrolytic and polydisperse nature of the material.
The full elucidation of the side chain structures and types of linkages
has not been achieved, in part because available anatytical methods have
not provided sufficient information. Due to the insolubI lity of lignin

sulfonates in organic solvents and their thermal instability, however,
methods such as NMR- and mass spectroscopy, extensively used In structural

studies of organic natural compounds, could not be applied. Our studies,


LOG wre Qgerces $0,/Cy

0.994 —Oe OXYGEN DEVIATION FROM 0.56 PER C
& (theoretically sulfonatable HIGH
Z 0.84 groups)
wy
° 0.74
%
a
a;
es aaieieeieeioend: . >--onkeeieeteeientenieniond waleted 0.
g 0.5
a 0.1lo
0.4
é 0.20
Z 0.3
5 0.30
a 0.24

LOW
0.14
$b ttt tt
11 (10 9 8 7 6 5 4 3 2
Fraction Number
FIGURE 17

OXYGEN FUNCTION IN ALSME DERIVATIVES CF MiL.

49

OXYGEN FUNCTION


(b)

(e)
CH=CH

CH=S03CH3

(d)

(a)
(c) (POLYMER)

OCH3
OAC

(b) (a) (POLYMER)

|

CH=CH
CH-S03CH3

(c) —_—_——-" (d)
OCH
OAC

FIGURE 19.

THE "REGENERATION PATHWAY" IN TERMS OF PEAKS, AS
VISUALIZED FROM THE SEPHADEX LH-20 FRACTIONATIONS.

59


EPA Review Notice

This report has been reviewed by the Environmental!
Protection Agency and approved for publication.
Approval does not signify that the contents
necessarily reflect the views and policies of

the Environmental Protection Agency, nor does
mention of trade names or commercial products
constitute endorsement or recommendation for use.


| |
—c-r- —- C-
| |
cH,0 oct,
O- Ae Ofc

DIMER EXPLANATION 1

ou OF ox
Fraction 1 - 5 Fraction 8,9
(6 7)

DIMER EXPLANATION 2

FIGURE 12.
HYPOTHETICAL DIMER FORMATION MECHANISM,

38


-1

&
o
iJ
©
5
a

H

2.
3

Fraction Number

°
'
10

NN c

sdnoag [Auoqueg yO 3U92U0) aAL2eLaY

a
‘g
o
oo 2 2 oO

om an nen ee oeee
ons ees mm * ~— + —_s Zt 2

AYLSUSZUT BALZLLIY

cm

1600

1800

FIGURE 11.

THE CARBCNYL REGION IN IR - SPECTRA OF FRACTIONS FROM SEPHADEX SEPARATIONS.


why its double bond should survive bromination, It seems as if this double
bond Is generated not only by conifery! alcohol endgroups as was assumed
hitherto.

The question as to the origin of the vinyl-groups Is belteved to be
answered only unsatisfactorily with coniferyl alcohol end-groups. tt could
be demonstrated that the most abundant [tntermedtate of the acidolysis reaction
pathway of the B-ether, as described by Adler, et.al., was able to forma
sulfonate with bisulfite fons (see Figure 21). This compound formed two
spots on TLC after acetylation with acetic anhydride and pyridine. Since
it also contained a carbonyl-group, its structure is expected to match com-
pound Il and acetylation forms the enolacetate II! and the keton II.

After transformation to ALSME-derivatives, 85% of the sulfonate appeared
as polymeric ether insoluble amorphous powder. The residual 15% ether-
solubles, showing eight distinct spots on TLC, exhibit an abnormal amount of
vinyl-groups in their NMR-spectrum (most of them conjugated to the aromatic
ring) and an acetyl-peak, the location of which matches an enolacetate.
Conclusions

LOMOLS seem to be a mixture of several particular mers and mer classes
that are in an equilibrium with each other and that rearrange and/or isomer-
ize easily to "regenerate" this mixture, The main reaction apparently involves
a double bond, which indicates vinyl polymerization as the mode of formation
of higher mers. Guaiacy|l-propene-2-sul fonic-acid-1 seems to play a key role
in this reaction. Its double bond stems obviously not only from coniferyl
alcohol endgroups.

It was found that hydrolysis products of B-ethers undergo sul fonatton,
and that these sulfonates are capable of forming vinyl groups via keto-enol
tautomerism, where the enol form polymerizes readily, probably tn a vinyl-

type polymerization.
62


SECTION I11

INTRODUCT I ON

Lignin is a polymeric substance or class of substances, composed of
phenol propane type units, which makes up about 20% to 30% of wood tissue.
Throughout the world, wood cellulose or wood pulp is produced on a very
large scale from woody tissue, leaving the lignins and lignin derivatives
in spent pulping liquor which is usually evaporated and burned to recover
heat and chemicals.

In one type of wood pulping process, the "sulfite" or "bisulfite
process," delignification is accomplished by the use of aqueous solutions
of metal bisulfites, either calcium, magnesium, sodium or ammonium bisul-
fite. Extensive investigations have been conducted and are continuing to
be directed to the development of effective and economically competitive
sulfite process recovery processes based on the recovery of effluent
liquors, and then evaporating and burning to recover useful process chemi-
cals and heat.

An alternative path is to develop methods for production of marketable
by-products from the lignin. From the sulfite process, lignin sulfonates
may be produced. Many studies have been conducted toward the objective
of producing useful products from these materials and success in applica-
tion and sales has been achieved in a few instances.

For further progress toward the aim of developing useful products,
two main advances seem to be necessary, i.e., firstly, to secure more
information concerning the physical nature and chemical structure and

reactions of the lignin sulfonates and the 30% of substances which coexist

5


HaGOH HC 0H

Ho Ho—
|

H COH H C=S0g

Oo
is)
=x
ao
an
oO
ny
oo
s
oO

OCH3 |
o~ o—
4 0/¢
"ea Scheme 4 30 + 1,803/Cg
CH=CH
CHSO3

O=0-2
be i= ol of
DS}
o
=

OCH3
S02H20 , OH
OCH3 CH=CH-CH2S03-
OH

{)

OCH3
Scheme 5 OH

OH

°
/
{=}
=
o
=
°o
/
oe

Scheme 6

FIGURE 16

48


Experimental Section

Ligntn sulfonates from a spent sulfite liquor were purified as usual
(cf, Section V) and fractionated by ton excluston chromatography. The LOMOLS
comprising part was neutralized, concentrated and freeze dried. The total
portion was transformed to ALSME derivatives in accordance with the proced—
ure described fn Section VI.

About half of the ALSME were fractionated over Sephadex LH-20 using
methanol! as mobile phase, The elution diagram exhibited a number of well
separated Tndividual peaks, the corresponding material of which were col-
lected and recovered by evaporation and drying under vacuum, The separation
had a very high degree of reproducibility. The substances from each indi-
vidual peak area were refractionated under the same conditions as used
before (see Figure 18). Whereas some peaks proved the high separation
potential of this method (peak d) other peaks exhibited elution diagrams
very similar to the total ALSME fraction (peak ¢). Material from corres-
pondtng mer classes (peak areas) was re-collected and recovered by evapora-
tion and drying. The oily residues were dissolved in a little methanol and
precipitated from ether 10:1. The ether solubles (ES) and the ether insolu-
bles (EIS) were recovered separately. The samples were analyzed by NMR
spectroscopy and elemental composition.

The other half of the ALSME derivatives was dissolved in chloroform,
and brominated under very mild conditions (temp. below 5-10°C) until there
was no more discoloration of the bromine. Then the material was recovered
and treated the same way as outlined above for unbrominated ALSME,

Veratry|-glycery!-f-arylether was submitted to a mild acid hydrolysis
(ACIDOLYSIS) following the procedure of Adler et. al. (30, 31) (0.2 N HCI in

dioxane:water 9:1; reflux for four hours), At that time, no more unreacted

55


The two monomeric’lignin sulfonates were isolated, crystallized and
identified as (3-methoxy-4-hydroxypheny | )-2-propene-|-sulfonate and I-
(3-methoxy-4-hydroxypheny|)-I-propene-3-sulfonate. The identity has been
verified by synthesis (1, 2).

Recent Studies

In more recent work, sodium lignin sulfonates from gymnosperm woods
were fractionated using Sephadex G-50 gel columns and eluting with distilled
water or with 107! to 1074 M NaCl solutions. Molecular weights of certain
lignin sulfonate fractions were estimated by ultracentrifuge sedimentation
equilibrium and were found to range in an orderly manner from about 70,000
in the mostly excluded initial eluates down to several hundred in the final
eluates. The sizes of the lignin sulfonate polymer molecules In terms of
equivalent Einstein spheres, ry were estimated from the measured molecular
weights and from intrinsic viscosities calculated using relationships
reported by Goring and coworkers. These ranged from about 7 to 70 R (3, 4).

For a lignin sulfonate of a particular molecular weight, Th was
estimated to be up to two times greater in water than in 0.1 M NaCl solu-
tion, behavior expected for an elastic network containing negative charge
sites in an environment of varying ionic strength.

We have shown that the concentration of sodium chloride in the aqueous
eluant exerts a significant influence upon the degree of success attained
in the fractionation of lignin sulfonates. To secure satisfactory repro-
ducibility in fractionation, the concentration of electrolyte must be held
constant. To secure optimum fractionation of lignin sulfonates, a par-
ticular level of eluant electrolyte concentration should be maintained
which is appropriate in relation to the pore size of the molecular sieve

material which is to be used.


EPA-660/2-74-069
dune 1974

STUDIES OF
LOW MOLECULAR WEIGHT LIGNIN SULFONATES

by
Wolfgang G. Glasser Bjorn F. Hrutfiord
Josef S. Gratz] Lennart N. Johanson
Kaj Forss Joseph L. McCarthy

duanita J. Collins

Roap/Task 21 AZX 60
Project 12040 DEH
Program Element 1BB037

Project Officer
Dr. H. Kirk Willard
Pacific Northwest Environmental Research Laboratory
National Environmental Research Center
Corvallis, Oregon 97330

for the

OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1.55


SECTION VII
PREPARATION OF ACETYL LIGNIN SULFONATE METHYLESTER
CALSME) AND THEIR SPECTROSCOPIC CHARACTERI ZATIONS

Introduction

The polyelectrolytic nature of lignin sulfonates has been the main
obstacle to their fractionation and characterization in the past. To
overcome the analytical! difficulties which were caused by this property,
we investigated the transformation of these compounds into derivatives
with properties required for structural studies, especially for NMR- and
mass spectroscopy. The synthetic basis for this transformation and the
analytical basis for the characterization of such derivatives had been
established before, using monomeric and dimeric lignin building units as
models. (See Section VI for details.) The transformation aimed at the
elimination of the polyelectric behavior by covering the polar groups:
hydroxy! groups by acetylation, and sulfonic acid groups by esterification.
Such acetylated lignin sulfonate methy! ester (hereafter called "ALSME")
would be sufficiently soluble in organic solvents to allow the applica-
tion of the more powerful analytical tools, such as NMR- and mass spec-
troscopy.

Transformation Procedure

The procedure used for the transformation of the LOMOLS-like monomers
and dimers was applied to lignin sulfonates with varying molecular weights
in a slightly modified form. The so-obtained ALSME products were found
to be soluble in chloroform, the solvent commonly used for recording

NMR-spectra.

29


SECTION XI]

CONCLUDING COMMENTS

Lignin sulfonates from the spent sulfite pulping liquor of Western
Hemlock have been separated in purified form using an alcohol extraction
and precipitation method as well as ion exclusion chromatography. Both
methods resulted in the isolation of mainly three fractions: high and
medium molecular weight lignin sulfonates, low mol. v. LS, and carbohy-
drate degradation products. In the alcohol extraction and/or precipita-
tion method, the separation was of lower quality than in the separation by
ion exclusion. Neither separation method, however, was capable of isola-
ting pure LOMOLS.

The characterization of lignin sulfonates worked best after covering
the highly polar hydroxy!l- and sulfonate-groups by acetylation and
esterification. NMR spectroscopy allowed a complete functional group
analysis and overall, appeared to be the most useful analytical method.

Summarizing the structural evaluation of low and high molecular
weight LS, I+ appears that LOMOLS are different from HIMOLS by configura-
tions that affect the color as well as surface tension properties. LOMOLS
were found to contain more phenolic hydroxyl-groups, more double bonds,
and probably more carbony!l=groups. The major type of linkage seems to be
one similar to a vinyl-polymer (two dimensional). This appears to be the
result of the sulfonation of acid hydrolysis products of alkyl-aryl-ethers.

Most properties of LOMOLS can be discussed in terms of this formation

hypothesis.

73


mobile solutes. The mobile solutes are mainly to be found in fraction

groups II - IV.
RED-SHIFT, nm
20--
I sie bit h4
LMNO
Pp
15
10
Sk
4
pc” ——— SPENT SULFITE LIQUOR
AA Oo ——— SULFONATED MWL
-
0 18 cl ! i 1
2200 3000 4000 5000 6000 7000 8000
EFFLUENT VOLUME, ml
FIGURE 3.

Shift of the Wavelength of the Absorption Maximum Near 280 nm
on Alkalizing Fractions of the Spent Sulfite Liquor and of
the Sulfonated Milled Wood Lignin Shown in Fig. 2.

Conclusions
From the presented results, it can be concluded that there are dis-
tinct differences between high- and low-, as well as between low~ and low

molecular wight lignin sulfonates. These differences are revealed by
gel permeation as well as thin layer chromatography and UV-spectroscopy.

Classification into four different groups (I-IV) seemed to be approximate.

17


RESEARCH REPORTING SERIES

Research reports of the Office of Research and
Monitoring, Envixonmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the ENVIRONMENTAL

PROTECTION TECHNOLOGY series. This series
describes research performed to develop and
demonstrate instrumentation, equipment and

methodology to repair or prevent environmental
degradation from point and non-point sources of
pollution. This work provides the new or improved
technology required for the control and treatment
of pollution sources to meet environmental quality
standards.


In sulfonation reaction in particular, the sulfonates do not have to

be separated from any carbohydrates. Control over all the organic material

fs guaranteed.
Experimental

Milled wood lignin, fsolated from Western Hemlock, was sulfonated
under mtld conditions c1o0°c, pH 1,5, 24h). The reactton product was
freed from excess SO, and sulfate ions. The calcium salts of the lignin
sulfonates-were fractionated over Sephadex G-25 in water. Twelve fractions
were collected and separately transformed to ALSMES.

These ALSMES were purified and investigated by NMR spectroscopy and
elemental analysis. The emptrical formulae were calculated for each of
the 12 fractions.

Results and Discussion

Representativeness of Milled Wood Lignin: The elution diagram of

Ca-LS from sulfonated MWL is compared with that of Ca-LS from a spent
sulfite liquor in Figure 13.

Differences between both elution diagrams can be visualized in the
very high- and the very low molecular weight region. Both differences
signify the peculiarity of the MWL preparation. The highest molecular
weight portion is absent since it cannot be extracted with neutral sol-
vents from the fiber. The lowest molecular weight material and the
extractives are missing as well, due to their removal prior to the isola-
tion of the wood lignin.

Structural Phenomena

By Elemental Composition; The empirical formulae for the Co-basis

were calculated for all of the twelve fractions. They are compiled tn

4|


TABLE 2
CHEMICAL SHIFTS FOR ACETYLATED AND ESTERIFIED
LIGNIN SULFONATES IN NMR SPECTROSCOPY ( 1-VALUES)

Region Location of Signals Type of Protons
2 2.00

3.72 aromatic H, except in sulfo-
nated coumarans,
a-vinyl-protons

3, 4 3.72 - 4.82 aromatic H in sulfonated
coumarans, .
8-viny!-protons

partially C,H

CyH-O-Ac (hon-sul fonated)
CaH-O-Ar (in coumarans)

5 4.82 - 7.50 Methoxy I-protons,
sidechain protons other than
in 3, 4 and 8
6 7,50 - 7.81 Aromatic acety|l-protons,

' except those ortho to bi-
pheny!-1| inkages

7 7.81 ~ 8.42 Aliphatic acety!l-protons,
aromatic acety!-protons ortho
to bipheny!-1inkages

8 8.42 - 9.62 highly shielded, mostly side-~
chain-protons

25


HYDROXYL GROUPS / AROMATIC PROTONS

0.5

0.33

Oo
\.
\,
\,
i— — NU
oN TTT ~e
~~ OCH, a—h—
NX ~
"0 oH
fee ee ~ eNe@ ee em me em eo —— = =
a a Sas 4—A—A
x ~ so
& phenolic on —_— “~, wo (8
~.U.07
T T T T T T T T T qv
13 12 11 10 9 8 7 6 5 4
Fraction Number
FIGURE 10.

CORRELATION BETWEEN PHENOLIC HYDROXYL GROUPS AND AROMATIC PROTONS IN
FRACTIONS FROM SEPHADEX SEPARATIONS.


therefore, were directed to establish modification procedures which
allow the conversion of lignin sulfonate fractions into derivatives with
the desired physical and structural properties by covering the polar
groups such as hydroxyl- and sulfonic acid groups without evoking
changes of the original side chain structures.

Another requirement of the transformation procedures to be applied
is their applicability to all the various lignin structures over the
entire range of molecular weights. To cover both the phenolic- and the
alcoholic hydroxy! groups as well, we applied the acetylation by pyridine/
acetanhydride.

To cover the sulfonic acid groups, the conversion to derivatives such
as acid chlorides, acid amides and esters was considered (13). Pre-
vious attempts to characterize acetylated monomeric lignin-type sul fochlor-
ides by mass spectroscopy, however, gave unsatisfactory results (14). As
a consequence, we explored the possiblility of establishing a suitable
method for esterifying the rather labile and reactive sulfonic acid groups
dominantly located in the a=position (benzyl-sulfonic acids) of lignin

side chains,

Synthesis

A variety of lignin related model compounds featuring pertinent side
chain structures and type of linkages were synthesized and sulfonated.
By following the briefly outlined transformation procedure (Figure 4) the
corresponding acetylated methy! sulfonates were formed’ (15, 16). The
obtained derivatives were soluble in organic solvents and purified by
recrystal lization and exhibited the physical properties required for NMR-

and mass spectroscopic analysis.

20


SECTION 11

RECOMMENDAT | ONS

The ion exclusion process for at least partial separation of lignin
sulfonates from sugars and other materials in sulfite spent liquors should
be developed further toward application on-at least a smal! pilot plant
industrial scale. The process desirably will be multi-stage and may be
conducted intermittently or batchwise using a column packed with an
appropriate fon exchange resin. Preferably the process should be carried
out using continuous fon exclusion resion handling equipment as soon as

such equipment becomes aval lable.

Further laboratory studies should be carried out using the now-reported
separation procedure for ALSME derivatives dissolved in organic solvents
based upon use of an appropriate molecular sieve materia! such as
Sephadex Hoo? and directed toward securing highly effective laboratory
separations of lignin sulfonates of differing molecular weights.
Simultaneously, further searches should be made to find organic

solvents which dissolve lignin sulfonates and permit direct gel
permeation chromatorgraphy separation. I+ seems possible that such
Procedures may permit the isolation in approximately pure form of

lignin sulfonates with degree of polymerization up to five or

possibly even up to ten.

Practially orlented research and development work should be ‘carried out
to learn how to make best economic use of the phenolic hydroxy! groupings
indicated to be present rather extensively in low molecular weight

lignin sulfonates. For example, a variety of ethers and esters could

be produced which may have industrial uses.

3


B-ether was left, as indicated by TLC. Without cooling, excess sodium
bisulfite in little water was added to the solution and reflux continued
for two days. Afterwards the solution was concentrated under vacuum and
redissolved in water. The aqueous solution was extracted several times
with plenty of chloroform to remove the chloroform solubles (unsul fonated
organics). About 2/3 of the starting material was recovered in the organic
phase. The rest (aqueous solution) was concentrated and run over an ion
exchange column in H-form to liberate the anions. The free acids were
freed from S05, neutralized, concentrated and freeze dried to yield mainly
the sodium salts of the sulfonated hydrolysis products of the B-ether.
The usual transformation (cf. Section V1) yielded the ALSME derivative
which was divided into an ether soluble and an ether insoluble part by
precipitation from ether. All steps were controlled by TLC, Sephadex G-25
and LH-20 chromatography, !R- and NMR-spectroscopy, as far as possible.
The elemental composition of the EIS ALSME derivative was determined and
its empirical formula calculated.
Results and Discussion

The refractionations were undertaken with the idea in mind to prove
the potential of isolating individual compounds from LH-20 separations.
This potential seemed to be very high, since the peaks exhibited were tall
and slender, indicating excellent separations (see Figure 18, 20). Also
NMR spectroscopy indicated good separation of the individual fractions.

However, upon refractionation most mers or mer classes did not exhibit
elution diagrams with single peaks, but demonstrated a rather pecul jar
instability, indicated in the "regeneration" of certain other peaks; as if

there was an equilibrium of the compounds represented by these peaks. By

57


SECTION X11

ACKNOWLEDGEMENTS

Dean R.W. Moulton, Chairman of the Department of Chemical Engineering,
University of Washington, provided helpful administrative assistance

for the project. Ms. June Demont did the secretarial work.

Professors K.V. Sarkanen and G.G. Allan made valuable suggestions

and their cooperation on several occasions Is appreciated.

The support of the project by the Office of Research and Monitoring,
Environmental! Protection Agency, and the help provided by Mr. George R.
Webster and Dr. H. Kirk Willard, the Grant Project Officer, is

acknowledged with sincere thanks.

75


sol
sot
40h
30
RELATIVE MIGRATION IN TLC
10-———— — — —— — — — —— —
ob \
4
oe eee
a5
toh
| COMPONENTS IMMOBILE ON TLUCEPLATES
x L 1 1 !
0° 7200 300d 7000 5000 6000 7000
A260 nm
BE
a i
| |
5r | 4
| |
rt Pouet dad
COMPONENTS IMMOBILE ON TLC-PLATES t | I
[| oer comer wie 1 piy | | i
3000 Taboo 000 TF ' subo | 6000 EFFLUENT VOLUME, ml
1 |
21 N01 9) 817) {5/413} 2 {

FIGURE

ELUTION DIAGRAMS OF

Above;

Below:
*TLC - Thin Layer Chromatography

CALCIUM LIGNIN SULFONATES

42

Secured from Spent Sulfite Liquor

Prepared from Milled Wood Lignin


Table 3. The phenolic- and aliphatic- acety! groups were determined by
NMR spectroscopy. The most essential functional groups were plotted in
diagrams (Figure 14). It is demonstrated that the content of phenolic
hydroxy! groups increases from high to low mol. w. material. The increase
seems to be steady in the high molecular weight range, exponential in the
low molecular weight range. The content of phenolic OH groups in MWL has
been reported to be about 0.3 per Cy unit. Apparently phenolic hydroxyls
are generated by the splitting of mainly a-arylether bonds (Figure 15);
however, the sharp increase of phenolic OH-groups in the LMWLS region
(fraction 6-1) cannot be explained only by this type of ether splitting,
but must involve some other reactions.

The. diagram of ether-OCH,-groups per Cy unit signifies this other
mode of phenolic OH group formation. The content of methoxy! groups remains
constant in the high molecular weight LS region, but drops off drastically
in the LMWLS region. This means that methoxy! groups are cleaved by the
sulfonation reaction under the formation of phenolic hydroxy! groups in
3-position (23). Sulfonate groups have been reported to increase steadily
for high molecular weight LS (24). This could not be verified. In con-
trast, the S-content of the various fractions as well as the content of
$0,-groups per Cy unit exhibits a bell-shaped curve with the culmination
point in fraction 6. Forss derived a lignin formulation from the fact
that the S-content for high molecular weight lignin sulfonates is steadily
increasing (25). He calculated that units of 10-18 Cy units share each
eight SO, groups. A stmilar calculation resulted in 15-20 Cg units for
every eight sulfonate groups in the same region of the elution diagram of

MWL.

44


in the sulfite process effluent liquors, and secondly, to secure more
information concerning how effectively and economically to separate these
components one from another.

Toward these general objectives, research has been continuing in this
laboratory over many years, and the studies now to be reported comprise a
continuation of this effort.

Earlier Studies

Since about 1945, research on spent sulfite liquor and lignin sul fon-
ates has been carried out at the University of Washington. During recent
years this research has been concentrated on lignin sulfonates and the
following specific work has been performed: Very low molecular weight
lignin sulfonates, previously shown to comprise a few percent of the tota
lignin sulfonates of gymnosperms, have been fractionated using the tech-
niques of column chromatography and multistage partition between butanol
and water. Two monomeric lignin sulfonates and a dimeric lignin sulfonate
have been isolated, crystallized, and shown to be the major components
of the lowest molecular weight fractions. Evidence has been obtained
for the presence of many other individual lignin sulfonates.

Dioxane lignin was prepared from carefully extracted wood and sul-
fonated. The resulting lignin sulfonates were separated into a high and
low molecular weight fraction, using a new.and fast method based on the
solubility of low molecular weight sulfonates in n-butanol saturated with
water. Upon chromatographic separation of the low molecular weight
material and purification of the appropriate fractions, crystalline
material was isolated and found to be identical with low molecular weight

lignin sulfonates previously isolated from spent sulfite liquor.