THE NATURE AND DISTRIBUTION OF EXTRACTIVES IN

Larix leptolepis GORD AND THEIR INFLUENCE ON

THE COLOUR OF REFINER GROUNDWOOD PULP

A thesis presented in part fulfilment of the requirements for the degree of

Doctor of Philosophy of the

University of London

by

Reginald James Orsler ARIC

Department of Botany Imperial College of Science and Technology London

May 1975

ABSTRACT

The present study originated from an appraisal of the problems

peculiar to the British Pulping Industry. Disc refiner

mechanical pulp is considered the most important product in

terms of future potential, and the effect that a mixed species

furnish has on the colour of this pulp is identified as a

major problem area. Japanese larch (Larix Zeptolepis Gord) is

introduced as the principal contributor to this dark colour,

and its extractives are selected as the most probable origin

of this colour.

A study of the nature of the heartwood extractives has been

carried out after removing these materials from the wood by a

succession of solvents in which there was a gradual change

from non-polar to polar character. The isolation and identifi-

cation of the individual constituents included the discovery of

two flavanones, not previously recorded for Japanese larch, and

the characterisation, for the first time, of a trilignol.

The distribution of the extractives within the lumina of the

wood elements, and the effect that the extracting solvents have

on this distribution has been investigated using scanning

electron microscopy. It is concluded from this, together with

the chemical examination, that the bulk of the extractives

reside within the cell walls of the wood. A further distribution

study, in which the wood elements were separated by dissection

and then extracted, has shown that the tracheid walls are the

principal site of extractives deposition. The possible

implications that these observations have on the theory of

heartwood formation is presented.

ii

Unextracted and extracted heartwood chips have been converted

to refiner mechanical pulp, and the colour characteristics of

the handsheets produced from these pulps have been studied.

This, together with an assessment of the effect of heartwood

extractives on sapwood pulp colour, and of the movement of

extractives during the pulping process, indicated that the

phenolic extractives are not removed during pulping and do

not have a great effect on the colour of the pulp.

iii

INDEX

Title page i

Abstract ii

Index iv

PART I GENERAL INTRODUCTION 1

1.1 The pulping of wood 1

1.1.1 Chemical pulping methods 2

1.1.1.1 The kraft or sulphate process 2

1.1.1.2 The sulphite processes 3

1.1.1.3 The semi-chemical processes 4

1.1.2 Mechanical pulping methods 4

1.1.2.1 Stone groundwood 4

1.1.2.2 Refiner groundwood 5

1.2 Pulping in the United Kingdom 6

1.3 Colour in pulp 8

1.4 Extractives and colour 13

1.5 Conclusion 15

PART II CHARACTERISATION OF THE EXTRACTIVES 18

2.1 Introduction 18

2.2 Heartwood extractives 20

2.2.1 Extraction 21

2.2.2 The acetone extract 22

2.2.2.1 Initial study 22

2.2.2.2 The chloroform soluble material 25

2.2.2.3 Ultraviolet spectral study 27

2.2.2.4 Tentative identification of flavonoids 32

iv

2.2.2.5 Separation of the flavanone mixture 33

2.2.2.6 Sephadex column separation 35

2.2.2.7 Chromatographic identification 37

2.2.3 The methanol extract 39

2.2.3.1 Initial study 39

2.2.3.2 Isolation of compound M 40

2.2.3.3 Preliminary tests 41

2.2.3.4 Experimental details of structural studies 43

i Alkali fusion 43

ii Spot tests 44

iii Acetylation 44

iv Rast molecular weight determination 46

v Nitrobenzene oxidation of compound M 47

vi GLC examination of nitrobenzene oxidation

product 48

vii Nuclear magnetic resonance studies 50

2.2.3.5 Discussion 51

2.2.4 The petrol extract 56

2.2.5 The ether extract 56

2.2.6 The water extract 57

2.2.6.1 Initial study 57

2.2.6.2 Examination of the hemicellulose fraction 58

2.2.6.3 Examination of the crystalline fraction 59

2.2.7 Quantitative estimation of the flavonoids in

the heartwood 59

2.2.7.1 Experimental 60

2.2.7.2 Results and discussion 60

2.3 Sapwood extractives 62

2.3.1 Experimental 62

2.4 Conclusions 63

PART III DISTRIBUTION OF EXTRACTIVES 67

3.1 Scanning electron microscopy 67

3.1.1 Introduction 67

3.1.2 Sample preparation 70

3.1.3 Photomicrographs 74

3.1.4 Discussion 100

3.1.4.1 Heartwood earlywood 100

3.1.4.2 Heartwood latewood 104

3.1.4.3 Sapwood 105

3.2 Cell wall distribution study 106

3.2.1 Introduction 106

3.2.2 Experimental 107

3.2.2.1 Preparation of tissue 107

3.2.2.2 Extraction 109

3.2.2.3 Measurement of optical density 110

3.2.3 Repeat experiment 110

3.2.4 Results and discussion 110

3.3 Conclusions 118

3.4 Mechanism of extractives' formation 120

PART IV REFINER GROUNDWOOD PULPING 126

4.1 Introduction 126

4.2 Main experiment 130

4.2.1 Experimental 130

4.2.1.1 Preparation and extraction of wood chips 130

4.2.1.2 Preparation of refiner groundwood pulp 132

4.2.1.3 Pre-treatments and preparation of pulp

handsheets 137

vi

4.2.1.4 Measurement of handsheet colour

characteristics 139

4.2.2 Results and discussion 145

4.3 Subsidiary pulping experiments 163

4.3.1 Pulping of larch sapwood 163

4.3.1.1 Experimental 163

4.3.1.2 Results and discussion 165

4.3.2 Movement of extractives during pulping 168

4.3.2.1 Experimental 169

4.3.2.2 Results and discussion 171

4.4 Conclusions 173

PART V CONCLUDING SUMMARY 176

Acknowledgements 186

References 188

vii

PART I GENERAL INTRODUCTION

1.1 THE PULPING OF WOOD

From the mid-nineteenth century, when Burgess and Watt in

England first produced a pulp from wood that was suitable for

papermaking, the conversion of wood into pulp for the manu-

facture of printing and packaging media has become one of the

most important industries of our modern society. In 1971 the

world production of paper and board stood at 131 million

tonnes, over twice the quantity produced just fifteen years'

before in 1956 (62 million tonnes) (Haas and Kalish 1972).

While this rate of increase may slow down a little with the

realisation that we have only a finite amount of wood avail-

able for pulp production, it is clear that even to maintain

this level of production the more efficient use of our forest

resources must be investigated.

While at first glance it would appear that wood pulp is a

single product, and that once produced may be used for any

paper or board material, further inspection reveals a much

more complex situation in which a variety of pulping methods

are used to produce special pulps for special end uses.

Broadly speaking there are two main methods used for pulp

production - chemical processes and mechanical processes -

although chemimechanical hybrid processes are also used.

1

The key to the pulping process lies in the middle lamella zone

of the wood's cellular structure. This has a high lignin

content and acts as the cementing substance, holding the wood

fibres together. In chemical pulping this zone is attacked by

chemicals and dissolved, thus allowing the fibres to separate.

In mechanical pulping the lignin is softened by heat (since it

acts somewhat like a thermoplastic) so that the fibres may be

separated by mechanical abrasion. In general the chemical

methods produce the high quality pulps, and the mechanical

methods the poorer pulps. A brief description of the current

methods in operation is useful; more detailed accounts may

be found in such standard works as Rydholm (1965), Casey

(1960) and MacDonald and Franklin (1969).

1.1.1 CHEMICAL PULPING METHODS

1.1.1.1 THE KRAFT OR SULPHATE PROCESS

This is the major pulping process today. Wood in the form of

small chips is treated ("cooked") at about 170°C with a

strongly alkaline liquor containing sodium hydroxide and

sodium sulphide. This results in a large proportion of the

lignin and some of the hemicelluloses being dissolved and

removed at the end of the cooking period with the spent

("black") liquor. The fibres can then be separated by a mild

mechanical treatment in a largely undamaged form. The

resulting pulp is brown in colour and is used in this form to

produce strong brown wrapping paper. Chemical bleaching will

produce strong, permanently white pulp which can be used for

a variety of purposes. The yield of fully bleached pulp is

2

about 40-43%, based on the weight of the wood used, as against

approximately 50% for the unbleached form. Softwoods are the

normal raw materials for this process, though some hardwoods

are also utilised.

Kraft cooking was originally carried out in batch digesters,

but the latest developments use continuous digestors through

which the chips travel slowly. The alkali is recovered and

re-used, and by-products such as turpentine are sometimes

collected. One unwanted by-product of this process is the

offensive odour which is due to the formation of organic

sulphur compounds such as mercaptans.

1.1.1.2 THE SULPHITE PROCESSES

The sulphonation and hydrolysis of lignin into a soluble

material using an inorganic sulphite salt has been used for

some considerable time. Originally the calcium salt was used

in the presence of a large excess of sulphur dioxide, but

more recently the sodium salts have become dominant as their

complete solubility allows a greater flexibility of cooking

conditions so that, for instance, resinous timbers can be

successfully pulped. Cooking temperatures are usually about

140°C. Two-stage cooking systems are in operation in which a

near neutral sulphite solution is used as a first stage,

followed by the addition of gaseous sulphur dioxide to pro-

duce an acidic liquor for the second stage cooking. In this

way high yields of light-coloured pulps can be obtained.

Yields are of the order of 45-60% and in the bleached form

3

such pulps are used for a wide range of printing and writing

papers.

1.1.1.3 THE SEMI-CHEMICAL PROCESSES

These part-chemical part-mechanical methods may be exampled by

the neutral sulphite semi-chemical process. Usually wood

chips are treated with sodium sulphite at about 170°C in a

continuous digester until just enough lignin has been removed

to allow mechanical defibration, with low power consumption,

when the chips are passed through a disc refiner (see also

under refiner groundwood process). The yield is about 70% and

the product can be used for corrugated packaging material.

Chemical bleaching lowers the yield to 50%-55% giving a white

pulp of suitable strength for book papers. This process is

particularly suited to hardwoods since hardwood lignin can be

easily attacked under these conditions.

1.1.2 MECHANICAL PULPING METHODS

1.1.2.1 STONE GROUNDWOOD

This is probably the oldest method of producing pulp from wood,

and the only one in which whole wood and not wood chips is

used. As the name implies the wood is ground to pulp by a

stone. Basically the grinder consists of a rapidly rotating

stone cylinder against which the bolts of timber are pressed,

with their longitudinal direction perpendicular to the

direction of rotation. Water is sprayed on to the wood where

it meets the stone in order to control the temperature, to

aid in plasticizing the wood, and to carry away the fibres and

fibre bundles which are abraded from the bolt's surface.

4

Since virtually none of the lignin is removed, yields are in

the region of 93%-98% of the original wood. However, the pulp

is comparatively weak and in the production of newsprint, for

which it is mainly used, a proportion of high quality chemical

pulp is added to increase the strength. Since full chemical

bleaching is uneconomic for this process, the spruces are the

preferred raw material as they give light-coloured pulps.

1.1.2.2 REFINER GROUNDWOOD

Arguably this process is the technique of the future, since it

is probable that much of the future's pulp will be produced by

a method based on this technique. Wet wood chips are fed into

the centre of a rotating metal disc system, involving either

two counter rotating discs or one static and one rotating

disc. A typical refiner would use two counter-rotating,

1.2 metre diameter, discs each driven by a 1000 hp electric

motor at speeds of between 1000 and 2000 rpm. The discs can

be as little as one-fifth of a millimetre or less apart,

their surfaces being toothed or grooved to facilitate the

abrasion of the chips. A great deal of research has gone into

the disposition of these grooves and teeth in relation to the

type of pulp required from the process.

The advantages of this technique are that the wood throughput

necessary to make the process economically viable is much

lower than for the other processes; and that there is a great

deal of flexibility of control over the end products. Mixed

species wood chips can be used and wood waste, even sawdust

5

can be incorporated into the intake. While the process is

mainly used for the production of board materials, it can be

used for newsprint manufacture.

Modern modifications have given rise to the "thermomechanical"

process, which is claimed to produce a stronger, less-damaged

pulp while using less energy for production. Here the

refining apparatus is enclosed in a steam-pressurised system

and the chips are fed into a steaming tube in which they are

softened by the combination of steam and heat before being

passed through the refiner. The pressure inside the system

blows the pulp out to the atmosphere after refining.

1.2 PULPING IN THE UNITED KINGDOM

The United Kingdom ptilping industry is summarised in table 1

(page 7). When one equates the number of pulp mills listed in

this table with the fact that the United Kingdom is one of the

world's largest producers and consumers of paper and board it

is apparent that the majority of our raw pulp, and paper and

board is imported. The six home mills produce about 10% of

our total pulp needs, but even with expansion this figure is

not likely to be increased since the total demand will also

increase. Accepting this situation, yet still requiring an

improvement in our home pulp production leads to the conclu-

sion that we must improve the quality of the pulp produced in

the United Kingdom. Thus less of the more expensive pulps

(and consequently more of the cheaper pulps) would have to be

imported resulting in a lowering of the total import bill.

6

Table 1

PULPMILLS IN THE UNITED KINGDOM (King and Smith 1972)

Mill Process Species used Quantity of

roundwood used (tons)

Products made from pulp

Ashton Containers Ltd Sudbrook Monmouthshire

Neutral sulphite semi-chemical

Homegrown mixed hardwoods oak, ash, alder, beech, etc.

120,000 ) Packaging

Bowaters Kemsley Mill Sittingbourne, Kent

Neutral sulphite semi-chemical

Homegrown mixed hardwoods 170,000 Packaging

Bowaters Kemsley Mill Sittingbourne, Kent

Stone groundwood

Imported spruce; some homegrown spruce

Newsprint

Bowaters Mersey Mill Ellesmere Port, Cheshire

Stone groundwood

Homegrown softwood logs,

mainly spruce, a little pine.

Newsprint

250,000

Bowaters Mersey Mill Ellesmere Port, Cheshire

Chip refiner groundwood

Homegrown softwoods New mill Probably newsprint

St Anne's Board Mill Co, Bristol Chip refine:. groundwood

Homegrown softwood spruce, pine, larch.

55,000 Paperboard

Thames Boardmills Workington Cumberland

Chip refiner groundwood

Spruce, pine, larch. 40,00D Paperboard

Scottish Pulp and Paper Mills Fort William Inverness

Chemical, two-stage sulphite

Spruce, pine, larch 270,000 ) Fine paper

In general the United Kingdom has a fragmented and hetero-

geneous forest geography such that it is not possible to set

up a large scale pulp mill supplied by a predominantly single-

species forest, as is the case in the United States of America,

Canada, and the Scandinavian countries. Therefore we have to

resort to a relatively low capital, low throughput system

capable of taking a mixed species wood intake. Of the avail-

able systems, only the refiner groundwood system satisfies

these requirements. Refiner groundwood pulp is a high yield

pulp (93%-98% of original wood), offering little scope for

further improvements in yield. However, the two other main

characteristics of a pulp can be considered. These are the

strength of the pulp and its colour.

Investigations into the possible methods for improving a

pulp's strength largely involve the study of the mechanics of

the process and the design of the grooved plates which face

the refiner discs, whereas the study of pulp colour is

basically a chemical problem. The work described in this

thesis is based on the premise that an improvement in the

quality of the pulp produced by the United Kingdom can be

effected by increasing the knowledge available on the origins

and nature of the colour in refiner groundwood pulps.

1.3 COLOUR IN PULP

The colour of a pulp is one of the main factors considered by

a potential user, for the colour and its stability are of

great importance, not only in the more obvious fields such as

8

book or writing papers, but also in the packaging industry where

the appearance of the container sometimes assists in selling the

product. Rather like the domestic detergents' industry, the

pulp industry is very sensitive to "whiteness" and "brightness"

(see footnote), and an almost imperceptible difference in colour

characteristics can make the difference between acceptance or

rejection of a pulp for a particular end use. However, this

sensitivity is ruled, to a certain extent, by economic consider-

ations which will become apparent when the methods used in

producing a white or bright pulp are discussed.

In figure 1 (page 10) the general compositions of wood and pulps

are expressed in histogram form. The efficiency of the deligni-

fying process, which is the basis of those methods involving

chemical treatments, can be seen clearly, but it can also be

seen that a small proportion of the original lignin still re-

mains. This lignin, particularly after the chemical

modifications it undergoes during the cooking period, is a

major cause of the dark colours of these pulps and must be

removed if a white pulp is to result. Since the chemical pulps

are high quality products, compared with the groundwood pulps,

and since only a small amount of lignin is to be removed,

comparatively expensive delignification agents can be used.

These usually involve treatment with strong oxidising agents,

such as chlorine or chlorine dioxide, followed by washing

with alkali to remove the chlorinated lignin.

Footnote: Whiteness is a measure of the amount of colour remaining in a material (pure white E no colour), while brightness is a measure of the amount of light reflected from a surface at a particular wavelength.

100 Wood

Hardwoods Softwoods

Groundwood and chip-refiner pulp

U

Extractives

EMS Lignin

Eftfa Hemicelluloses

EIDII Cellulose

B Bleached

U Unbleached

Neutral Sulphite _semichemical pulp

rn co

50

0-

Wood

&

Two - stage Bisulphite pulp

Sulphite Sulphate pulp (Kraft) pulp

U

U

1

Typical yields and compositions of commercial wood pulps

100

4., 50

yi

f---- i Book Corrugated Paperboard Bags, papers packaging Newsprint and Wrappers,

board, etc. Building boards. Newsprint.

Bags, Newsprint.

E Strong wrapping papers, sack, etc.

High quality writing, Printing, Photographic papers.

The chemical composition and end-uses of various woodpulps. (After Packman 1965)

By contrast the groundwood pulps rely on their high yield and low

cost of production for their viability. Consequently a bleach-

ing technique which is expensive or which removes a large

proportion of the pulp's substance cannot be considered.

Groundwood pulps are usually brightened rather than bleached

either by an oxidising or a reducing agent that modifies, but

does not remove, the lignin. The oxidants may be exampled by

sodium and hydrogen peroxide, and the reductants by sodium bi-

sulphite, sodium dithionite and zinc dithionite. These chemicals

are relatively cheap, and do not place too much of a burden on

the total cost of the groundwood process.

In order that the groundwood pulps may be considered for roles

that until now have been the preserve of the bleached chemical

pulps, their final colour must be made whiter and more stable.

In countries where the raw material for groundwood pulp is

mainly the light-coloured softwoods, eg spruce (Picea spp),

this problem is associated with the chemistry of lignin since

it is this fraction of the pulp substance that provides most of

the colour. However, in the United Kingdom some darker-coloured

softwoods are also pulped. These are not liked by the pulp

producers, but they have to be accepted since the high demands

placed on home-grown timber by the pulp mills cannot be met by

supplying spruce alone. A typical intake for a refiner ground-

wood pulp mill in the United Kingdom would be 50% spruce, 25%

larch (Larix spp), and 25% pine (Pinus spp). The larch fraction

would contain the darkest-coloured timber and would be made up,

predominantly, of Japanese larch (Larix leptolepis Gord syn

11

L. kaempferi. Sarg).

The effect that these timbers have on the colour of a pulp can

be better appreciated with some knowledge of how the colour

characteristics of a pulp are expressed. Brightness is usually

expressed in terms of the amount of light that is reflected from

the surface of a pulp mat compared with that reflected from a

compacted tablet of magnesium oxide powder. For brightness re-

lated to whiteness the light used has a wavelength of 457 nm,

produced by a standard blue filter, since the dominant shade for

most pulps is yellow and because the human eye is particulary

sensitive to yellow. With magnesium oxide giving 100% reflec-

tivity, the fully bleached chemical pulps will give about 98%

while the darkest pulps will give only 10%. The reflection

characteristics of a pulp throughout the visible light spectrum

can be determined by using a series of colour filters. It

should also be noted that the human eye cannot discern degrees

of brightness with equal ease from 0% to 100%. Variations at

the extreme ends of this scale are difficult, sometimes impos-

sible, to detect and it is in the central part of the scale that

small differences in measured reflectivity are seen as marked

differences by the observer.

In a study of the production of refiner groundwood pulp from

various homegrown species, Packman (1967) found that the bright-

ness of Sitka spruce pulp was 62% while that of Scots pine and

Japanese larch (the darkest of those tested) were 56% and 38%

respectively. It is clear that at these levels of reflectivity,

where the human eye is most alert to change, the inclusion of

12

quantities of larch into a refiner groundwood pulp will have a

considerable detrimental effect on the colour of the resultant

pulp. In a further study of the production of refiner groundwood

pulp from Japanese larch (Anon 1971) it was shown that the heart-

wood was the main contributor to the colour of the whole wood

pulp. It was found that for a whole tree which gave a pulp of

brightness 40%, the sapwood pulp had a brightness of 54% while

the heartwood pulp had a brightness of 38%. Since the composition

of the cellulose and lignin in the sapwood and heartwood are,

from a practical viewpoint, similar it would seem reasonable to

assume that the cause of the larch pulp's dark colour can be

found in its extractive's composition.

1.4 EXTRACTIVES AND COLOUR

"Wood extractives" is a general term referring to all those

materials that can be removed from wood by extraction with

neutral solvents without altering the wood's basic structure.

As they do not usually make any contribution to the mechanical

properties of wood they have also been described as the extrane-

ous or secondary constituents of wood. However, it should be

appreciated that this basic definition of wood extractives is by

no means rigorous, for there are materials in some woods that

are virtually impossible to extract without altering the wood's

structure, or that require strong alkali to remove them; they

are, nevertheless, referred to as extractives. Between species,

the extractive content can vary from almost nothing to over

one-third of the wood's weight, and it can include a wide variety

of different classes of organic compound. The polyphenolic

extractives are probably the most ubiquitous, but fats and

resins, terpenes, tropolones, alkaloids, and carbohydrates also

13

occur in timber.

The qualitative composition of the extractive fraction of any

timber species is characteristic of that species and may be used

for chemotaxonomic purposes (Swain 1963) provided that it is

realised that the various parts of the tree (eg sapwood, heart-

wood, bark, leaves, roots) can have different characteristic

extractives. However, a quantitative survey of the extractive

composition within a single species will show that the amount of

each individual component can vary from tree to tree and even

within a single tree. It is generally accepted that within the

trunk of a tree the heartwood contains the major proportion of

the extractives and that the outer heartwood is particularly

rich in extractive materials.

The great variety of attractive colours and tones apparent in

the utilised timbers of the world can be attributed to the

variety of extractives found in them. This is understandable

when one considers the nature of these extractives in relation

to the more fundamental reasons for colour production. Colour

will be conferred on a molecule through its ability to absorb

part of the visible spectrum of light, and it can do this if it

contains a chromophore or colour-producing group. These chromo-

phores are usually unsaturated, ie they contain multiple bonds,

and it is the mobility of the electrons associated with these

bonds and their ability to absorb energy that produces the

colour. There are also certain groups which will deepen or

darken the colour produced by a chromophore and these are known

as auxochromes. Simple conjugation, ie alternate double and

14

single bonds, can produce colour although a single isolated

double bond is not sufficient to do this, eg CH2 = CH2 is

colourless, but CH3 (CH = CH)6 CH3 is yellow.

From the point of view of wood chemistry the most important

chromophore is probably the carbonyl group (› = 0) and the most

important auxochrome is the hydroxyl group (-OH). These, in

combination with conjugation, are probably responsible for most

of the colour produced by the wood extractives, and can be

exampled by the orange dalbergiones (Eyton et al 1966) and the

yellow xanthones and flavonols (Karrer 1958) whose basic struc-

tures are represented by I, II, and III respectively.

0 II

I

0 III

1.5 CONCLUSION

It would seem clear from the preceding discussion that the

colour of the pulp produced from Japanese larch in the United

Kingdom's refiner groundwood mills is one of the major obstacles

15

to the upgrading of home-produced mechanical pulp, and that an

investigation into the role that the larch extractives play in

the production of pulp colour would be of value. Japanese larch

has been shown to contain phenolic extractives that can affect

the colour of chemically produced pulps (Migita, Nakano and

Toroi, 1951; Fujii 1952). However, no similar study has been

carried out on mechanically produced pulp from this species.

In the following dissertation an investigation into the nature

of Japanese larch phenolic extractives is described, together

with a study of their distribution in the wood at cell levels.

This latter point is important in that processes for the removal

or modification of interfering extractives can be dependent on

their point of deposition in the wood. The de-resination of

pulp is an example of this. The resin, which would normally

create pitch problems in paper production, is often found to

occur only in the ray cells. These are much smaller than the

tracheids and can be mechanically removed by sieving.

The work is concluded with a description of some pulping

experiments in which the effects of the identified extractives

on pulp colour is assessed.

While the thesis as a whole is based on the need for a practical

industrial problem to be solved, it also provides information

on some of the fundamental aspects of the bio-synthesis of

wood extractives and the formation of heartwood. These subjects

have been covered in a recent review (Hillis 1972) in which it

16

is clear that further work is necessary if the mechanisms of

these complex processes are to be resolved.

17

PART II CHARACTERISATION OF THE EXTRACTIVES

2.1 INTRODUCTION

Hasegawa and Shirato (1951) and Nishida, Ito, and Kondo (1952)

obtained a colourless compound from the heartwood of Japanese

larch which they found to be identical to distylin, a compound

previously isolated from Distylium racemosum by Nishida and Kondo

(see Kondo 1951).

Migita, Nakano and Toroi (1951), and later Fujii (1952) described

a flavanone derivative from Japanese larch heartwood which caused

difficulties in the sulphite pulping of this timber, and which

they found to be identical to taxifolin, earlier isolated from

Douglas fir (Pseudotsuga menziesii syn P. taxifaia) by Pew

(1948). Kondo and Furuzawa (1953) in assessing the importance

of the extractives in the utilisation of Japanese larch timber,

separated distylin and a further flavonoid, katuranin.

These results were rationalised in studies on European larch

(Larix decidua) by Gripenberg (1952) in which he demonstrated

that the material isolated by Hasegawa and Shirato,and which

they identified as distylin, was, in fact, a mixture of taxifolin

and aromadendrin, this latter compound being identical to Kondo.

and Furuzawa's katuranin.

Taxifolin or distylin is also known as dihydroquercetin, since

it is a derivative of quercetin, but is properly termed

3,3',4',5,7-pentahydroxyflavanone (I). Similarly aromadendrin

or katuranin is also known as dihydrokaempferol, and is properly

termed 3,41 ,5,7-tetrahydroxyflavanone (II).

18

HO OH I R = -OH

II R = -H

OH 0

Brewerton (1956) studied the acetone extracts of both European

and Japanese larch, and provided what is still probably the

most complete description of the phenolic heartwood extractives

of these timbers. Using the technique of preferential disso-

lution in various solutions and solvents, together with

cellulose absorption chromatography, he found that for Japanese

larch, dihydroquercetin and dihydrokaempferol constituted 73%

and 15% respectively of the total acetone extract, which itself

was equivalent to 4.5% of the dry weight of the wood. Most of

the remaining 12% he classified generally as phlobaphenes,

tannins, and a solvent soluble lignin. In addition he isolated

small quantities of a third crystalline material which he con-

sidered to be an hydroxyflavanonol, though he was unable to

identify it.

More recently the Japanese workers Demachi, Terezawa and Sasaya

(1968) have reported the isolation of the yellow compounds

quercetin and kaempferol from the heartwood, as well as the

related flavanonols.

Apart from the phenolic extractives, the genus Larix is excep-

tional among the conifers (softwoods) in that it contains

comparatively large amounts of water-soluble hemicelluloses

which consist mainly of arabinogalactans. Bouveng and Lindberg

(1958) showed that the arabinogalactans found in the larches

19

were of two types, to which they designated the letters A and B.

Aspinall (1964) and Aspinall, Fairweather and Wood (1968) found

both arabinogalactan-A and -B present in Japanese larch, and

determined the structure of arabinogalactan-A.

The composition of Japanese larch resin has received scant

attention. Stairs (1968) carried out a study on the oleoresin

of several larch species. Dealing specifically with the mono-

terpene fraction, he found that Japanese larch yielded a mixture

comprising approximately 80% a-pinene, with smaller quantities

of a-pinene, limonene, camphene, and myrcene. Mills (1973) in

a more extensive study of the Larix oleoresins concentrated on

the diterpenes and showed that for Japanese larch the main con-

stituents of a complex mixture were thunbergol, (epi)-manool,

isopimaric acid, and abietic acid.

In the following examination of the heartwood extractives of a

British grown Japanese larch tree, it was considered that the

phenolic constituents are those most likely to be responsible for

the inherent colour or colour change of larch groundwood pulp,

and so they received the most attention. Only a brief appraisal

of the terpenaceous and polysaccharide fractions of the

extractives has been undertaken.

2.2 HEARTWOOD EXTRACTIVES

Thin layer chromatography (TLC) was used to monitor all

operations in this section. Unless otherwise stated the system

used consisted of ready-prepared silica gel plates (Merck) with

a benzene/dioxan/acetic acid 90:25;4 mixture as the developing

20

solvent. Phenols were detected either by spraying the plates

with diazotised sulphanilic acid, or by fuming the plates with

ammonia and then spraying with anhydrous ferric chloride in

ethanol.

Gas liquid chromatography was carried out using a Pye Unicam

Series 104 gas chromatograph fitted with a flame ionisation

detector.

Ultraviolet and infrared spectra were obtained using Unicam

SP800 and SP200 spectrophotometers respectively. Nuclear

magnetic resonance (nmr) spectra were provided by

Dr D M X Donnelly of Dublin University using a Perkin Elmer

60 MHz instrument.

Carbon and hydrogen quantitative analyses were carried out by

Dr F B Strauss of Oxford, and group quantitative analyses by

the Alfred Bernhardt Laboratories of West Germany.

Melting points were determined using sealed evacuated Pyrex

tubes.

Solutions were evaporated to dryness under reduced pressure by

rotary evaporator. Solution temperatures did not exceed 50°C

during this operation.

2.2.1 EXTRACTION

The heartwood from a single Japanese larch tree, grown in the

south-east of England, was converted to sawdust while in the

21

"green" (undried) condition, and immediately freeze dried.

This latter operation was carried out using an Edwards Vacuum

Freeze-Drying Unit, Model 30 PIT (figure 2, page 23). Having

determined its moisture content, the sawdust was spread out on

shallow aluminium trays, sealed in the unit, and brought to

-10°C. The unit was then evacuated to give a vacuum of

tig 2 Vacuum freeze-drying unit. Edwards High Vacuum, Model 30 PIT

at Rf 0.24 predominated. Since it appeared unlikely that this

mixture could be separated satisfactorily in one chromato-

graphic step, the total extract was treated with a series of

solvents in an attempt to produce fractions containing fewer

constituents than the original. Four fractions were obtained,

including the residue, after treating the extract successively

with boiling 100-120 petroleum ether, boiling carbon tetra-

chloride and cold diethyl ether.

The petrol soluble portion (3.9% of the total acetone extract)

did not contain any phenolic material. Submitting it to column

chromatography on alumina produced a series of colourless and

pale yellow oils, but no crystalline material. Nothing further

was done with this fraction.

The carbon tetrachloride soluble portion, which comprised only

1.3% of the total acetone extract was shown, by TLC, to contain

a variety of phenolic compounds, most of which had Rf values in

the range 0.4-0.8. No further studies on this small fraction

were made.

The ether soluble portion (19.4% of the acetone extract) and the

residue (75.3% of the acetone extract) were found to be of

similar composition. Examination by TLC showed that both

fractions contained four phenolic compounds, the main con-

stituent occurring at Rf 0.24 with the other three, in order of

decreasing spot size, at Rf 0.39, 0.32 and 0.49. Results

obtained in a comparison with known compounds from the Princes

Risborough Laboratory (PRL) extractives collection are detailed

24

in table 2 (page 26). From this it would appear that the

compound at Rf 0.24 (Al) is probably dihydroquercetin and that

that at Rf 0.39 (A3) is probably dihydrokaempferol. The remain-

ing two compounds do not appear to be quercetin or kaempferol

since they differ from these two known compounds in both Rf

values and colour reactions.

In an attempt to obtain a quantity of more pure compound Al,

a portion of the ether soluble fraction was boiled with chloro-

form according to the recrystallisation procedure for

dihydroquercetin recommended by Brewerton (1956). On filtering

and leaving to cool the solution did not deposit any crystals.

However TLC examination of this solution and the undissolved

residue showed that compounds A2 and A4, together with the

majority of compound A3 and some Al had been dissolved by the

chloroform, leaving a residue of compound Al with a little

compound A3. This residue was dissolved in boiling ethanolic

water, decolourised with charcoal, and allowed to recrystallise

to give creamy white needles, melting point 239-242°C undepressed

by the addition of dihydroquercetin.

2.2.2.2 THE CHLOROFORM SOLUBLE MATERIAL

The chloroform solution was evaporated to near-dryness and in

this more concentrated form shown to contain five different com-

pounds. The additional compound, which was phenolic, had an

Rf of 0.63 on TLC, but was present in only trace amounts.

Developed thin layer chromatograms of this five component

mixture were submitted to a series of reagents normally used to

25

Table 2

TLC EXAMINATION OF THE ETHER SOLUBLE FRACTION OF THE ACETONE EXTRACT USING SILICA GEL COATED PLATES

AND BENZENE/DIOXAN/ACETIC ACID 90:25:4 AS ELUTING SOLVENT

Sample or Standard Rf

Spot Colour

Under visible light Under visible light after

fuming with ammonia Under ultra-violet light after fuming with ammonia

Ether soluble fraction compounds

Al 0.21 Pale grey P violet/red Yellow

A2 0.32 Pale grey P violet -

A3 0.37 Pink Blue Yellow

A4 0.48 Pink - Yellow

Dihydroquercetin 0.21 Pale grey P violet/red Dark yellow

Dihydrokaempferol 0.38 Pink Blue Yellow

Quercetin 0.30 Yellow Brown/yellow Yellow

Kaempferol 0.41 Yellow Yellow Yellow/brown

•

classify flavonoid compounds and phenols, and the results may

be seen in table 3 (page 28). Indications from this, and the

previous table, are that the compounds Al-A4 inclusive appear

to be flavonoid in nature, although the precise group to which

they belong could not be ascertained. The fifth compound did

not give a positive reaction to any of these reagents, either

because it was not a flavonoid compound or because it was

present in too small a quantity to be visible after reacting.

2.2.2.3 ULTRAVIOLET SPECTRAL STUDY

In order to obtain further information on the four flavonoid

compounds, the characteristics of their UV spectra were examined

according to the procedure laid down by Mabry, Markham and

Thomas (1970). This type of study has the advantage that only

small amounts of material (0.1 mg or less) are required for

analysis. Sufficient material was obtained from one 20 cm x

20 cm preparative TLC plate coated with a one mm thick layer

of silica gel on which the chloroform soluble portion had been

loaded. After development the plate was fumed with ammonia and

viewed under UV light so that the areas containing compounds

Al-A4 inclusive could be marked. These areas were scraped from

the plate and each resulting powder sample extracted twice with

3 ml aliquots of ethanol (UV pure ethanol was used throughout

this exercise). The solutions were filtered from the silica

powder by reduced pressure through a glass sinter.

The UV study entailed determining the UV spectrum between 190

and 450 nm of each compound and then measuring the effect that

a series of standard reagents have on this spectrum.

27

Table 3

SPOT COLOURS OBTAINED WITH VARIOUS SPRAY REAGENTS ON A DEVELOPED TLC PLATE OF THE CHLOROFORM SOLUBLE MATERIAL

SILICA GEL PLATE; BENZENE/DIOXAN/ACETIC ACID 90:25:4 DEVELOPING SOLVENT

Compound FeSO4/Na2CO3 (aqueous)

Fume with NH3 then alcoholic FeCl3

Na2CO3 (aqueous)

AlC13 in alcohol A1C13 in alcohol under ultra-violet

light

Al Blue/black Blue/black Yellow Yellow Dark yellow

A2 Blue/black Blue/black Yellow - Pale yellow

A3 - Red/brown Yellow Yellow Dark yellow

A4 - . Red/brown Yellow - Pale yellow

Each solution obtained from the extraction of the TLC powder

was further diluted so that the optical density of the major

absorption peak was between 0.6 and 0.8. This was the stock

solution from which the following spectra were obtained:-

1 The stock solution.

2 The stock solution in the cell after treatment with three

drops of sodium ethoxide solution. This spectrum was

re-run after 5 minutes to check for flavonoid decomposition.

The sodium ethoxide solution was prepared by adding

cautiously 2.5 g freshly cut metallic sodium to 100 ml

ethanol.

3 The stock solution in the cell after treatment with six

drops of a 5% solution of anhydrous aluminium chloride in

ethanol.

4 The solution from (3) after the addition of three drops

dilute hydrochloric acid (50 ml concentrated hydrochloric

acid plus 100 ml water).

5 The stock solution in the cell after being shaken with

excess coarsely powdered anhydrous sodium acetate. The

spectrum was re-run after 5-10 minutes to check for

decomposition.

6 The stock solution in the cell after treatment with five

drops of boric acid solution (ethanol saturated with.

anhydrous boric acid) followed by saturation with

coarsely powdered anhydrous sodium acetate.

The wavelengths of the peaks and shoulders obtained from these

spectra are recorded in table 4 (page 30).

29

Table 4

ABSORPTION WAVELENGTHS (nm) OBTAINED IN UV SPECTRAL STUDY OF FLAVONOID COMPOUNDS

CompomdUntreatedSodimenmddeAlminimcIdorideA1C1,MC1 o

Sodium acetate Boric acid/

Sodium acetate

290 248 sh 313 313 289 sh 293 Al 333 sh 323 375 small 372 330 329

288 245 sh 290 290 sh 288 290

A2 329 sh 324 308 sh 306 326 325

367 small

292 ---'-' 248 312 312 294 294 A3

331 sh 328 366 small 370 small 328 327

288 245 291 283 sh 288 288

At 328 sh 325 309 sh 305 325 325

366 sh 366 small

sh = shoulder

small = minor peak

All four compounds had similar absorption characteristics in

the range studied. The shape of the spectral curve obtained

with the stock solutions was typical of that described for

flavanones and flavanonols, ie a main peak in the range

270-295 nm (designated band II) with a shoulder or low inten-

sity peak in the range 320-340 nm (designated band I). These

two flavonoid classes cannot be differentiated by UV methods,

since the saturated C2-C3 bond prevents the detection of the

3-hydroxyl group. This same saturated bond also precludes the

possibility of gaining information on the hydroxylation pattern

of the B-ring. However, the hydroxylation pattern of the A-ring

can be explored.

After treatment with sodium ethoxide, all four compounds gave

a spectrum exhibiting a bathochromic shift of 33-37 nm and an

increased intensity for band II. This is indicative of the

A-ring containing a 5,7-dihydroxy group. Sodium acetate also

produced a bathochromic shift (36-40 nm) for all four compounds,

again indicative of a 5,7-dihydroxy disposition. The sodium

acetate/boric acid spectra did not differ from the sodium

acetate spectra, suggesting that no ortho-dihydroxy groups were

present. The similarity between the aluminium chloride spectra

and the aluminium chloride/hydrochloric acid spectra also

suggests that no ortho-dihydroxy groups are present. However,

when compared with the stock solution's spectra, the aluminium

chloride/hydrochloric acid treatment did produce a bathochromic

shift of 17-23 nm consistent with the presence of a 5-hydroxy

group.

31

The information gained from this spectral study suggests that

all four compounds are based on one of two structures: the

3,5,7-trihydroxyflavanone or the 5,7-dihydroxyflavanone, ie

HO 0

OH 0

HO or

2.2.2.4 TENTATIVE IDENTIFICATION OF FLAVONOIDS

Barton (1968) has described a spray reagent that will detect

3-hydroxyflavanones on TLC. The developed plate is sprayed

with a suspension of zinc dust in acetone, dried, and then

sprayed with 6N hydrochloric acid. Bright red spots appear

coincident with the 3-hydroxyflavanones. This reagent, when

used on a developed TLC plate loaded with the four flavonoid

compounds together with dihydroquercetin and dihydrokaempferol

standards, gave red spots for the two standards, and for the

two flavonoid compounds, Al and A3, thought to be dihydro-

quercetin and dihydrokaempferol. The remaining two flavonoid

compounds, A2 and A4, did not react, and may thus be considered

to be flavahones not having a 3-hydroxy group.

The dihydrokaempferol standard taken from the PRL extractives

collection and used throughout this study had been isolated

from the heartwood of coigue (Nothofagus dombeyi), and it was

noticed that an impurity in this sample had the same Rf value

on TLC, and the same colour reactions, as compound A4. In

studying coigue, Pew (1948) found dihydrokaempferol associated

with its equivalent flavanone naringenin. Running a naringenin

32

standard (from the PRL extractives collection) and the chloroform

soluble mixture on TLC showed that the standard and compound

A4 both had an Rf of 0.49, and reacted identically to the spray

reagents listed in tables 2 and 3 (pages 26 and 28 respectively).

It would appear likely, therefore, that compound A4 is naringenin.

The relationship between dihydrokaempferol and naringenin can

be seen by the several similarities in their response to phenol-

and flavonoid-detecting spray reagents (see table 2, page 26,

and table 3, page 28). By the same criteria a similar relation-

ship can be assumed to exist between dihydroquercetin and

compound A2. The flavanone equivalent to dihydroquercetin is

eriodictyol, and a sample of this compound, kindly provided by

Professor Geissman of the University of California, was

compared with the flavonoid mixture as described above for

naringenin. Compound A2 and the eriodictyol standard showed

identical reactions to the spray reagents and had identical Rf

values.

In concluding this section, it would appear that virtually the

whole of the acetone extract is made up of four flavanone

compounds, dihydroquercetin, which predominates, dihydro-

kaempferol, eriodictyol, and naringenin. Their isolation would

enable confirmation of their identity.

2.2.2.5 SEPARATION OF THE FLAVANONE MIXTURE

Where dihydroquercetin and dihydrokaempferol occur together in

nature they have been found to be extremely difficult to

separate as they crystallise together in various proportions.

33

In the present case, the inclusion of two closely related

flavanones in the mixture adds to the separation problem.

While paper chromatography and preparative TLC could be used,

they would be tedious for collecting comparatively large

quantities and it was felt that amongst current separation

techniques there would be a simpler and quicker method of

resolving this problem.

Dry column chromatography (Loev and Goodman 1967) using

silica gel with the benzene/dioxan/acetic acid 90:25:4 eluent

system proved unsuccessful as did a conventional polyamide

column using a methanol/water eluent in which the methanol

content was gradually increased as elution progressed. A

cellulose column, constructed and used as described by Brewerton

(1956), was not completely successful, as indeed was reported

by Brewerton, although the column did remove the bulk of the

dihydroquercetin. The resulting mixture of dihydrokaempferol,

eriodictyol, and naringenin, contaminated with a little

dihydroquercetin was used for subsequent separation attempts.

Sephadex, a cross-linked dextran, has found wide application

in the field of gel filtration, mainly with aqueous solutions.

However, the introduction of a lipophilic derivative, Sephadex

LH-20, has extended the use of this technique to organic sol-

vent systems. Such a system, using methanol as the eluent,

proved suitable for the separation of the flavonoid mixture

(see footnote).

Footnote: After this work was completed, it was found that Johnston, Stern and Waiss (1968) had published a short note on the separation of flavonoid compounds by column chromatography using Sephadex LH-20.

34

2.2.2.6 SEPHADEX COLUMN SEPARATION

100 g Sephadex LH-20, after soaking for 24 hours in methanol,

was used to produce a column 70 cm long and 3 cm in diameter.

Approximately 0.5 g of the flavonoid mixture eluted from the

cellulose column was dissolved in 5 ml methanol and introduced

on to the column. Elution by methanol at a rate of 1 ml min-1

was allowed to continue for 24 hours. The eluate was collected

in 10 ml aliquots using an LKB automatic fraction collector.

Fraction collection began immediately after the mixture was

placed on the column, with the tubes being numbered accordingly.

The tubes were monitored by TLC for the presence of flavanones

although the tubes containing these compounds were indicated by

the appearance of crystals at the tube's mouth when the solvent

had evaporated a little. It was found that tubes 53-55

inclusive contained dihydrokaempferol alone, tubes 57-61

inclusive contained a mixture of naringenin and dihydro-

quercetin (naringenin predominated in tube 57 and dihydro-

quercetin in tubes 60 and 61, but the bulk of these two com-

pounds were found in the same tubes), and tubes 65-69 inclusive

contained eriodictyol alone.

Tubes 53-55 inclusive were combined in an evaporating basin

and the methanol allowed to evaporate at room temperature.

The crystalline residue was recrystallised from hot water to

give whitish crystals, melting point 233-235°C undepressed by

an authentic sample of dihydrokaempferol.

35

Tubes 57-61 inclusive were combined and the methanol allowed

to evaporate at room temperature. The residue was taken up

in a little acetone and loaded on to two preparative TLC plates

(1.0 mm thick coating of silica gel; developing solvent

benzene/dioxan/acetic acid 90:25:4). After development the

solvent was allowed to evaporate at room temperature and the

plates were fumed with ammonia which allowed the bands of

separated compounds to be clearly seen when viewed under ultra-

violet light. The silica gel coating containing the naringenin

band was scraped off the plate and extracted with methanol.

The dihydroquercetin was not recovered. The yellowish residue

obtained by evaporation of the methanol solution was recrystal-

lised from aqueous methanol to give crystals contaminated with

a yellow amorphous material. Careful treatment with hot

diethyl ether dissolved the crystals only, which were subse-

quently recovered, after filtration, by evaporation of the

ether. Recrystallisation from aqueous methanol gave needles,

melting point 251-253°C undepressed by an authentic sample of

naringenin.

Tubes 65-69 inclusive were combined and the methanol allowed

to evaporate at room temperature. The residue was recrystal-

lised from ethanolic water to give a pale yellow microcrystal-

line powder. The mother liquors were centrifuged off and the

powder washed with water which was also removed by centri-

fuging. After drying, the powder gave melting point 264-

267oC undepressed by an authentic sample of eriodictyol.

36

In order to obtain a purified sample of dihydroquercetin,

approximately 0.5 g of the chloroform insoluble, ether

soluble, portion of the acetone extract was put through the

Sephadex column using the same technique as described pre-

viously. This successfully separated the dihydrokaempferol

from the dihydroquercetin, the latter, after recrystallisation

from water, yielding white needles melting point 241-243°C,

undepressed by an authentic sample.

2.2.2.7 CHROMATOGRAPHIC IDENTIFICATION

In order to provide additional proof of identity, the isolated

flavanones, together with authentic samples, were subjected to

three different TLC systems. In all cases the flavanones

reacted in precisely the same manner as their authenticated

counterparts.

System 1:- Silica gel plate (Merck prepared) with benzene/

dioxan/acetic acid 90:25:4 as eluent.

System 2:- Silica gel plate (Merck prepared) with

chloroform/ethyl acetate/formic acid 35:55:10

as eluent.

System 3:- Cellulose plate (Merck prepared) with chloroform/

methanol/formic acid 80:10:15 as eluent.

After development plates were fumed in ammonia and sprayed

with ethanolic ferric chloride. This gave blue/black spots

for dihydroquercetin and eriodictyol and red/brown spots for

dihydrokaempferol and naringenin. Rf values are given in

table 5 (page 38).

37

Table 5

TLC Rf VALUES FOR FLAVANONE COMPOUNDS

Compound System 1 System 2 System 3

Dihydroquercetin 0.19 0.65 0.44

Eriodictyol 0.32 0.76 0.70

Dihydrokaempferol 0.34 0.76 0.73

Naringenin 0.45 0.80 0.88

38

2.2.3 THE METHANOL EXTRACT

2.2.3.1 INITIAL STUDY

The crude methanol extract, which consisted of 102 g as a dark

brown syrup, was treated with a succession of solvents in an

attempt to produce a series of fractions with each containing

fewer constituents than the original extract.

The whole extract was first repeatedly washed with aliquots of

acetone until it appeared that no further dissolution was

taking place. The resulting insoluble residue was dried in a

vacuum desiccator, and the acetone solution evaporated to dry-

ness. The residue from the evaporation step was treated with

ethyl acetate, using the same procedure as that described above,

to produce an insoluble fraction and a residue from the evap-

oration of the ethyl acetate solution. Similar treatment of

the latter residue with diethyl ether yielded an ether insol-

uble and an ether soluble fraction. Thus a total of four

fractions were obtained:-

a Acetone insoluble: 1.15 g dark brown powder

b Ethyl acetate insoluble: 9.45 g dark yellow powder

c Ether insoluble: 10.3 g pale buff powder

d Ether soluble: 4.7 g pale buff powder

Each fraction was submitted to the TLC examination using

dihydroquercetin, dihydrokaempferol, eriodictyol, and naringenin

as standards. After treating the developed plates with the

ammonia/ferric chloride reagent, fraction (d) was seen to con-

sist mainly of dihydroquercetin, with some dihydrokaempferol,

much less eriodictyol, and a trace of naringenin. Fractions

(c) and (b) also contained these compounds in the same relative

order of abundance, although in much smaller amounts. The

major part of fractions (c) and (b) appeared to be associated

with a streaked spot extending from the origin to about Rf 0.2.

Fraction (a) remained entirely at the origin.

Excluding fraction (a), which was not studied further, it

appeared that the methanol extract was made up of a mixture of

the flavanones identified in the heartwood acetone extract,

together with appreciable amounts of another material hereafter

referred to as compound M.

2.2.3.2 ISOLATION OF COMPOUND M

Since it was known that the four flavanone compounds detected in

the methanol extract were all soluble in diethyl ether, fractions

(b) and (c) were combined and repeatedly extracted with boiling

diethyl ether. This removed a small amount of the flavanone

mixture, but was by no means complete in its action. The ether-

insoluble material was dissolved in methanol and boiled with a

few grams of decolourising charcoal. The hot solution was

filtered off (No 50 Whatman paper) and the cooled filtrate

poured into a relatively large quantity of cold, stirred, diethyl

ether. The flocculent precipitate which formed was filtered

off on a Buchner funnel and dissolved in the minimum amount of

methanol. This methanol solution was slowly passed through a

glass column packed with polyamide (Woelm, 45 cm x 2.5 cm) which

had been thoroughly washed with methanol prior to its use.

This process resulted in the bulk of the colouring material

being retained on the column. After concentration, the eluate

was put through a second polyamide column of similar dimensions

to the first.

40

The resulting solution, a clear golden colour, was evaporated

to near-dryness and left in a refrigerator for several days in

an attempt to promote crystallisation. However, this did not

occur and the methanol solution was poured into a large volume

of diethyl ether to precipitate the solid. After filtration on

a Buchner funnel, and drying in a vacuum desiccator, compound

M (14.4 g) appeared as a non-crystalline, bulky solid, pale

buff-orange in colour.

The efficiency of the separation of compound M from the flava-

nones was checked by TLC using freshly prepared plates coated

with polyamide (Merck) and methanol/acetic acid/water 90:5:5

as the eluent. The developed plates revealed the position of

the compounds after fuming in ammonia followed by spraying with

ferric chloride in ethanol. Compound M showed as a single pale

blue/black spot at Rf 0.7 whereas dihydroquercetin and dihydro-

kaempferol, as standards, appeared as dark blue/black spots,

both at Rf 0.45.

2.2.3.3 PRELIMINARY TESTS

Compound M did not give a colour (red or yellow) when dissolved

in ethanol and treated with magnesium and hydrochloric acid,

thus indicating that it was probably not a flavonoid compound.

It-dissolved readily in dilute sodium hydroxide solution to

produce a rich-brown colour, and it reacted with ethanolic

anhydrous ferric chloride solution to produce a blue/black

colour indicative of a phenolic group being present. Gentle

heating of compound M with Tollen's reagent produced a silver

mirror, suggesting the presence of a reducing group (eg an

41

aldehyde). The ultraviolet spectrum of compound M in UV-pure

% 1 ethanol showed an absorption peak at 203 nm. (EI cm = 663), a

1 shoulder at 232 nm, and a second peak at 281 nm.

(E1° cm 97)

indicating aromatic (benzenoid) centres.

The compound remained amorphous in spite of repeated attempts

to produce a crystalline form, and it lacked a precise melting

point. A melting point determination resulted in the powder

sintering at 130°C to a much-reduced volume of pale red-brown

material. At 156°C this changed to a frothy mass, in which

form it remained, slowly assuming a dark brown colour, until

290°C when the heating was stopped.

The infrared spectrum of compound M, prepared as a nujol mull,

gave absorption peaks at 3440, 1604 (with a shoulder at 1638),

1520, 1270, 1220, 1139, 1084, 1034 cm-1, with small peaks at

. 860, 820 and 720 cm

-1. Of these, the large peak at 3440 cm-1 is

most probably due to the stretching frequency of the hydroxyl

group, and those at 1604 and 1520 cm-1 due to aromatic skeletal

vibrations. The remaining absorption peaks are difficult to

assign precisely and are better regarded as a "fingerprint"

pattern peculiar to the compound under consideration.

Carbon and hydrogen microanalyses carried out on compound .M gave

61.71% carbon and 6.06% hydrogen (for C301134011 carbon = 63.14%

and hydrogen = 6.01%)

1+2

2.2.3.4 EXPERIMENTAL DETAILS OF STRUCTURAL STUDIES

i ALKALI FUSION

1 g compound M, 2 g potassium hydroxide, 2 g sodium hydroxide,

and 1 ml water were thoroughly mixed together in a boiling tube.

The air was flushed out with nitrogen and this atmosphere main-

tained while the tube was immersed in a silicone oil bath,

heated slowly up to 200°C and held at that temperature for ten

minutes. At and above 120°C the dark yellow mixture frothed and

became more viscous until, finally, a rubber-like mass separated

from the dark-brown liquor. After cooling, the mixture was

diluted with water, acidified with dilute hydrochloric acid,

and extracted with ether. The yellow/brown ether solution,

after extraction with sodium bicarbonate solution, was washed

with water, dried with anhydrous sodium sulphate, and evaporated

to dryness to yield 0.1 g phenolic syrup. The sodium bicarbon-

ate solution was acidified with dilute hydrochloric acid and

extracted with ether. This ether solution was washed and dried

as before, and then evaporated to dryness to yield 0.2 g acid

material.

The phenolic and acidic components were studied separately by

TLC and seen to contain, in both instances, a complex mixture of

individual compounds. The separated phenolic fraction, when

sprayed with diazotised sulphanilic acid revealed several bright

red spots which faded extremely rapidly. In addition, none of

the simple phenols run as markers (phenol, resorcinol, catechol,

pyrogallol) matched precisely the individual components in the

developed mixture. Such a complex array with no predominating

components, was reminiscent of the alkali fusion products of

lignin preparations, and a Japanese larch dioxan lignin from the

43

PRL collection (for preparation see Browning 1967) reacted

similarly when submitted to alkali fusion. No further work was

done on the alkali fusion products.

ii SPOT TESTS

In order to confirm the suspected lignin-like character of com-

pound M it was submitted to the phloroglucinol/hydrochloric acid

test. This gave a deep purple (positive) colour, indicating

the presence of a lignin-like compound containing a cinnamalde-

hyde end-group (Adler et al 1948).

In addition, compound M and its acetate were submitted to a spot

test which is specific for aldehyde groups (Dickinson and

Jacobsen 1970). A purplish colour (positive reaction) was

obtained when either of the two compounds (about 20-30 mg) was

added to a solution of 4-amino-3-hydrazino-5-mercapto-1, 2,

4-triazole (100-200 mg) in 2 ml. 1N sodium hydroxide, and the

resulting solution aerated for about one minute.

iii ACETYLATION

0.2 g compound M, 2 ml pyridine, and 2 ml acetic anhydride were

placed together in a small stoppered flask and left at room

temperature for two days. The resulting solution was poured

into about 100 ml water, with stirring, when a precipitate

formed. The precipitate was filtered off and dried in a vacuum

desiccator, after which it was dissolved in a little hot

methanol. Hot water was slowly added to the methanol solution,

which was kept at near boiling point, until the solution acquired

a slightly cloudy appearance. It was then allowed to cool, when

an off-white flocculent precipitate appeared. The precipitate

Ls. Li

was filtered off and dried as before and then dissolved in a few

millilitres of benzene. After treatment with decolourising

charcoal, the filtered benzene solution was poured into a

relatively large volume of 60-80 petrol to produce an off-white

precipitate which was filtered from the solution and dried under

vacuum to give 0.25 g fine, off-white, powder.

TLC indicated that the acetate was a single substance. Plates

coated with silica gel, when developed with benzene/dioxan/

acetic acid 90:25:4 showed the acetate as a single spot at

Rf 0.67, and when developed with ethyl acetate as a slightly

tailed single spot at Rf 0 41. In both cases the position of

the spot was revealed by spraying the plate with antimony

pentachloride in carbon tetrachloride (20% v/v) followed by

heating to 105°C.

When submitted to a melting point determination, the acetate

did not appear to have a sharp melting point. The sample

changed slowly from a sintered solid to a frothy viscous mass

over the temperature range 85-93°C. Continued heating resulted

in the material finally becoming a free-moving, clear, light

brown liquid at approximately 125°C.

Carbon and hydrogen microanalyses showed the acetate to contain

61.65% carbon and 5.86% hydrogen (for C40H44016 carbon = 61.53%

and hydrogen = 5.68%). The methoxyl content was found to be

11.01%, the acetyl content 29.88%, and the C-methyl content

10.83% (for a trimethoxy, penta-acetoxy compound with the

empirical formula C40H44016 the figures are 11.92%, 27.57%, and

9.63% respectively).

45

The infrared spectrum produced from a chloroform solution of the

acetate revealed that all the hydroxyl groups had been

acetylated (ie the large absorption peak at 3440 cm 1 found for

compound M had disappeared) and that a large absorption peak had

appeared at 1740 cm-1 due to the carbonyl bonds in the acetyl

groups. Other peaks for the acetate occurred at 3000, 2993,

1663, 1600, 1510, 1462, 1420, 1368, 1230, 1190, 1145, 1117, and

1030 cm-1 with small peaks at 900 and 850 cm-1.

iv RAST MOLECULAR WEIGHT DETERMINATION

This was carried out on both compound M and its acetate.

Approximately 5 mg of the compound under test and 50 mg of

camphor were weighed accurately into a small ignition tube

(7 mm x 50 mm) which was subsequently sealed in a gas flame.

The sealed ignition tube was submerged in a silicone oil bath

and the whole heated slowly up to 200°C when the contents were

converted to a melt.

After cooling, the tube was broken open and a melting point

determination carried out in triplicate on the melt, using

sealed, evacuated Pyrex tubes. A similar determination was

carried out on the pure stock of camphor. The molecular weight

was then determined using the formula:

M = wM'(K - d) Wd

where w = weight (g) of the compound under test

M' = molecular weight of camphor

46

K = 40 000 molecular weight of camphor

d = depression of camphor melting point in cIC

induced by the addition of the compound

W = weight (g) of camphor taken.

The molecular weight determination for compound M was not

without problems, since it was found to be impossible to produce

a clear melt by heating the compound and camphor together.

The result must, therefore, be considered suspect since it is

quite possible that not all of compound M was mixed intimately

enough with the camphor to affect its melting point. However,

no such problem was encountered with the acetate and the

determination of its molecular weight proceeded without com-

plications. The two molecular weights, as determined were:-

Compound M 445

Acetylated compound M 785

v NITROBENZENE OXIDATION OF COMPOUND M

25 mg compound M, together with 4 mis 2N sodium hydroxide and

0.5 ml nitrobenzene were sealed in a small nickel bomb and

heated for three hours at 160°C.

The cooled reaction mixture was transferred to a separating

funnel using both water and diethyl ether. The ether layer was

removed and the aqueous layer repeatedly washed with ether to

remove the nitrobenzene. The resultant aqueous layer was made

just acid with dilute hydrochloric acid and then extracted with

ether.

47

Having been dried with anhydrous sodium sulphate, the ether

solution was evaporated to dryness to yield a yellow/brown oil

which was examined by TLC and by GLC (see page 20).

The developed plate from the TLC separation was fumed with

ammonia and sprayed with alcoholic anhydrous ferric chloride to

reveal a series of brownish spots, one large and predominating,

the remainder small. Vanillic acid and vanillin, run as stan-

dards on the same plate showed that the large spot corresponded

with vanillin at Rf 0.60, and that vanillic acid (Rf 0.51) might

be present as one of the three small spots centred at Rf 0.52.

The remaining unidentified small spots had Rf values of 0.66,

0.43 and 0.36.

vi GLC EXAMINATION OF NITROBENZENE OXIDATION PRODUCT

The GLC apparatus was fitted with a 90 cm (3 ft) glass column,

0.6cm (2; inch) outside diameter, packed with diatomite C 80-100

mesh (acid washed and dimethyldichlorosilane treated) carrying

5% DC11 silicone oil. This was maintained at 170°C with the

carrier gas (nitrogen) flowing at 60 ml min-l. The detector

oven was maintained at 250°C.

The nitrobenzene oxidation product was silylated before being

injected into chromatograph. This was achieved by using

N,0-bis (trimethylsilyl) acetamide (BSA), a very reactive

silylating agent (Klebe et al 1966). The total oxidation

product (less the small amount used for TLC examination) was

dissolved in 1 ml pyridine and transferred to a small specimen

bottle. 1 ml BSA was added to this and the tube capped and

48

placed over phosphorus pentoxide in a desiccator. The mixture

was left at room temperature for two hours, after which time it

was injected directly into the chromatograph using a 10 pl micro-

syringe. Retention time values were used to identify the

separated components, and a comparison of peak heights was used

to assess the relative quantity of each component present.

In GLC analysis, the passage of a separated constituent through

the detector is signified by a change in the signal voltage from

the detector. This signal is passed through an amplifier and

recorded as a peak on a flat-bed recorder. The elapsed time

between injection and peak appearance (retention time) is

characteristic of the compound associated with the peak, and

the peak height (strictly the area under the peak) is directly

proportional to the amount of compound passing through the

detector.

Initial injection of the silylated oxidation product showed that

at least ten compounds were present with retention times of

0.95, 1.75, 2.60, 3.45, 4.7, 6.05, 6.35, 7.6, 9.2 and 13.25

minutes. However, after adjusting the amplifier to give just

less than full-scale deflection on the recorder for the major

peak, only one peak remained of significant size. A comparison

of peak heights showed that it constituted 95% of the total

oxidation product, and its retention time (1.75 minutes) corres-

ponded exactly with that of a sample of silylated vanillin.

49

vii NUCLEAR MAGNETIC RESONANCE STUDIES

Nmr spectroscopy is one of the most powerful tools at the

disposal of the structural organic chemist. Its most common

use lies in its ability to recognise functional groups and

molecular fragments in a molecule, and to quantify the

distribution of protons between these groups. The spectra

are usually determined from a solution of the material under

test, but since the technique relies on a property of the protons,

the solvents used must not contain such entities. Hence

deuterated solvents are widely used.

Initial attempts to secure a nmr spectrum of compound M were not

very successful due to its insolubility in most of the solvents

normally used for such determinations. Deuterated pyridine

proved the most satisfactory solvent for this compound, but even

this produced a poor spectrum which only indicated the presence

of ',CHOH and - CH2O - groups.

Nimz (1966) has produced some extremely good nmr spectra of

lignin hydrolysis fragments, using the acetates rather than the

original compounds. The advantage of using this derivative is

that it is commonly soluble in chloroform, the deuterated form

of which is the preferred solvent for nmr spectroscopy.

Additionally, all hydroxyl groups, which contain one proton,

are replaced by acetyl groups, which contain three protons,

thus making for a larger signal.

Consequently the acetate of compound M, dissolved in deuterated

chloroform, was submitted to nmr spectroscopy. The resulting

50

spectrum was not as clear as was hoped for, largely because the

smaller peaks were partially obscured by a noisy base-line signal.

However, using a recent review by Ludwig (1971) on the magnetic

resonance spectra of lignins, and the conclusions made by Nimz

(1966), the peaks were characterised. This is presented in the

first three columns of table 6 (page 52).

2.2.3.5 DISCUSSION

Preliminary work on a sample of compound M isolated by a

relatively simple procedure indicated that the compound was

possibly a derivative of dihydroquercetin (eg a glycoside),

until confusing results prompted a reappraisal of the purity

of the sample. It became evident that dihydroquercetin had a

great affinity for compound M, and remained in trace amounts

after what was thought to be a reasonable extraction procedure.

However, the isolation procedure outlined in the text (page 40)

satisfactorily overcame this problem.

Initial spot tests on the isolated material gave some indication

of its chemical character, showing it to contain aromatic

centres, and to have a phenolic nature and reducing properties.

The lack of crystalline form and of a sharp melting point

suggested a possible polymeric or high molecular weight compound.

With such compounds it is usual to attempt some form of degra-

dation reaction in the hope that the molecular fragments thus

formed may help in identifying the whole molecule. At this

early stage of the investigation alkali fusion seemed appropriate.

This is one of the classical methods of degrading organic

compounds and finds wide application. In many instances it has

51

Table 6

DATA FROM NMR SPECTRUM OF ACETATE OF COMPOUND M

T value (ppm)

Group responsible Relative peak area

No of protons for trimeric structure

8.07 Aliphatic acetoxyl 12.1 12

7.82 Aromatic acetoxyl 3.8 3

6.27 Methoxyl 8.8 9

5.1-6.0 Propane side chain protons 7.4 8

4.0 Vinyl? 1.5 2

3.04 Aromatic protons 9 9

provided valuable information in the study of natural products.

That the alkali fusion produced such a complex mixture was

disappointing, since the isolation and identification of all

fragments would have been a major undertaking. Nevertheless

the very fact that it gave such a multitude of degradation

products, together with their reaction with the TLC spray

reagent hinted at its possible lignin-like nature. This was

confirmed by the phloroglucinol/hydrochloric acid test which

indicated that it was a lignin-like compound containing a

cinnamaldehyde end-group. The existence of an aldehyde group

in both compound M and its acetate was confirmed by the

application of a spot test specific for aldehydes.

A reappraisal of the ultraviolet spectrum for compound M showed

that it conformed to the basic shape produced by lignin-like

molecules (Goldschmid 1971). The infrared spectrum was

compared with those exhibited by various lignin preparations

(Hergert 1971), between which there were seen to be broad

similarities. However, apart from confirming that compound M

was an hydroxylated and aromatic compound, no more precise

information could be gained from these spectra.

Nitrobenzene oxidation of lignin compounds provides one of the

most useful methods of assessing the basic structure of such

molecules (Chang and Allan 1971). The procedure converts a

proportion of the lignin into identifiable aromatic fragments,

which are mostly a mixture of aldehydes. The major represen-

tatives are vanillin, syringaldehyde, and p-hydroxybenzaldehyde

53

which derive from guaiacyl, syringyl, and phenyl centres

respectively in the original lignin molecule. That compound M,

when submitted to this technique, gave vanillin as 95% of its

oxidation product and did not give identifiable quantities of

syringaldehyde or p-hydroxybenzaldehyde, strongly indicated

that the original molecule is comprised entirely of guaiacyl

units.

The molecular weight determinations for both compound M and

its fully acetylated derivative indicated that the molecule is

small compared with normal lignin polymers. Freudenberg (1964)

has suggested that such small lignin-like molecules should be

referred to as lignols. This nomenclature was adopted for

compound M.

For a lignol composed of guaiacyl units, and giving positive

reactions to tests for phenolic and cinnamaldehyde groups, the

assignment of a tentative basic structure is possible if

cdgnisance is taken of the formulation for softwood lignin

proposed by Freudenberg and Harkin (1964) and Freudenberg (1965).

Thus:-

TH2OH CH2OH HO

CH.CH—O r.CH-0

OH OH OMe OMe

CH=CH.CHO

54

For n = o (ie for a dimer) the molecular weight takes the value

374 and for n = 1 (the trimer) 570. Thus the value, as deter-

mined by the Rast method (445), falls between the dimer and

trimer molecular weights. This situation was not unexpected from

the practical difficulties encountered in attempting to

determine the molecular weight of compound M. However, the

determined molecular weight for the fully acetylated derivative,

785, is very similar to the calculated value for the fully

acetylated trimer, 780.

The nmr spectrum obtained for the acetate included most of the

groups present in the tentative trilignol structure (table 6,

page 52). It can be seen that the relative peak area associated

with each group does not correspond precisely in every case with

the proton distribution in the tentative structure. In addition,

the peak representing the aldehyde proton, which is strongly

deshielded and should occur in the range T-2.0 to t0.25, was not

observed, possibly because the noisy base-line signal was

sufficient to obscure a peak due to a single proton. Nevertheless,

in considering the nmr spectrum as a whole, broad general

agreement existed between the size and disposition of the peaks,

and the proton distribution in the suggested trilignol structure.

The quantitative carbon and hydrogen microanalyses obtained for

both compound M and its acetate can be used collectively to

indicate a trimeric structure, although the acetate's results

conform more closely to this than do those for compound M. The

additional results obtained for the acetate, ie the acetyl,

methoxyl, and C-methyl contents, suggest a penta-acetoxy

55

trimethoxy compound rather than any other configuration for a

compound of this type and having a molecular weight of

approximately 780.

2.2.4 THE PETROL EXTRACT

The petrol extract, which consisted of 10.2 g thin yellow/brown

oil, was only briefly examined for the presence of crystalline

components. None were found.

Alumina column chromatography produced a series of near-

colourless syrups, all of which failed to crystallise, even

after standing for some considerable time.

Thin layer chromatography, using silica coatings and benzene

as the eluant, showed that the extract consisted of at least

twelve compounds. There were four major components, each one

showing as a blue fluorescent spot under ultraviolet light,

at Rf

values 0.78, 0.25, 0.09 and at the origin. After

spraying the plate with antimony pentachloride in carbon

tetrachloride (20% v/v), followed by heating to 105°C these

spots became visible as different colours - Rf 0.78 yellow/

brown, 0.26 blue, 0.09 brown, and the origin orange.

2.2.5 THE ETHER EXTRACT

This tiny fraction, only 2.8 g pale brown syrup from over

2 kg wood, was examined by the standard TLC method. Using the

four flavonoid compounds found in the acetone extract as

markers, the developed plate showed that the ether extract was

mainly dihydroquercetin, together with traces of dihydrokaempferol

56

and eriodictyol. No naringenin was detected. A series of

unidentified faint spots with high Rf values could indicate

that simple phenols are present in this extract in trace

amounts.

2.2.6 THE WATER EXTRACT

As has been noted previously (page 19) the water extract of

Japanese larch consists mainly of hemicelluloses, of which the

arabinogalactans should predominate. Although it is unlikely

that these hemicelluloses affect the colour of the system

under study, the extract was examined.

2.2.6.1 INITIAL STUDY

Practical problems, such as frothing and its attempted