New The sampling and identification of components in volatile … · 2014. 11. 25. · Techniques for the sampling of volatile organic mixtures in the atmosphere necessitate the concentration

THE SAMPLING AND IDENTIFICATION OF COMPONENTS

IN VOLATILE ORGANIC MIXTURES

by

R.B. Chesterman, Utp. App. Chem.

Submitted in partial fulfilment

of the requirements for the degree of

Master of Science

UNIVERSITY OF TASMANIA

HOBART

December 1982 cp-A-ce_rect. Maid— (183)

Except as stated therein, this thesis

contains no material which has been accepted

for the award of any other degree or diploma

in any university, and that, to the best of my knowledge

and belief, the thesis contains no copy or paraphrase

of material previously published or written by another

person, except when due reference is made in the text.

R. B. Chesterman

ACKNOWLEDGEMENTS

I wish to gratefully acknowledge the advice and

encouragement of my supervisors, Professor H. Bloom,

Dr. B.V. O'Grady, and Dr. I.R.C. Bick, who offered many

helpful suggestions during the course of this work.

I am also indebted to Mr. C. Richards of the Chemistry

Department workshop for the construction of the prototypes

of - the sample desorption equipment; and to Mrs. H. Hen,

Mrs. B. Dix and Mrs. B. Thompson of the secretarial staff

who assisted with the preparation of the diagrams and

figures, and who answered once again with patience,

questions concerning preparation of the text that must

have been put to them many hundreds of times before.

The assistance of the staff of the Central Science

Laboratory for help with mass spectral and information

processing of material is gratefully acknowledged.

The help of Dr. J. Madden, and the staff of the CSIRO

Forest Research Section at Cambridge, for logistic and

other support is .. gratefullyacknowledged. For the help of

my wife Lynne with the typing, and her understanding of the

curtailment of family life associated with part-time

studies I will be always grateful.

CONTENTS

Page No.

CHAPTER I INTRODUCTION. 1

CHAPTER II EXPERIMENTAL TECHNIQUES FOR THE CONCENTRATION AND ANALYSIS OF VOLATILE ORGANIC COMPOUNDS. 2 A.Concentration and Trapping Techniques. 3 B.Direct Headspace Collection. 15 C.Separation and Analytical Techniques. 17 • D.Detection and Analytical Techniques. 25 E.Sources of Error in Trace Volatile

Analysis. 29

CHAPTER III DEVELOPMENT OF A SAMPLING SYSTEM FOR VOLATILE ORGANIC MIXTURES. 32 A.Selection of a Sample Adsorption Medium. 34 B.Development of Sample Collection

and Desorption Equipment. 40 C.Blank Determinations and Sample

Trap Storage. 47

CHAPTER IV AN EXPERIMENTAL COMPARISON OF VOLATILE ORGANIC SAMPLING TECHNIQUES. 54 A.Experimental. 55 B.Discussion. 66 C.Summary and Conclusions. 69

CHAPTER V VOLATILE COMPONENTS OF SOME NATIVE PLANTS. 70 A.Components of the Steam Volatile Oil

of Drimys lanceolata. 71 B.Components of the Steam Volatile Oil

of Prostanthera lasianthos 83 C.Volatile Components of

Boronia megastigma 94

CHAPTER VI VOLATILE COMPONENTS OF PINUS RADIATA. 102 A. The Composition of the Steam Volatile

Components of Bark Oil P. radiata 106 B. Collection of Headspace Samples From

the Bark of P. radiata. 110 C. A Comparison of Bark Volatiles From

a Girdled P. radiata 113 D. Composition Changes of Bark Headspace

of P. radiata. 122

CHAPTER VII CONCLUSIONS

126

REFERENCES

129

APPENDIX

143

ABSTRACT

Public awareness of the toxic effects of some volatile

organic compounds present in the working and home

environment has created a requirement for the accurate

measurement of the concentration of these substances.

Similar techniques may be used for the examination of the

volatile components of plant material and assist in

determining the optimum economic time for harvesting, and

also provide useful information in the areas of plant-insect

interaction. This thesis examines some of the sampling and

analytical aspects for the determination of trace volatile

organic compounds present in air.

A brief critical review of the literature is presented

describing concentration and trapping techniques, headspace

collection, separation and analytical techniques, detection

and measurement of the separated components and the sources

of error in trace volatile analysis.

The development of an experimental sampling system for

volatile organic compounds in air, based on trials conducted

with activated carbon and a porous polymer is presented.

• Two methods of sample desorption are described and the

development of sampling equipment based on Tenax GC, thermal

elution with secondary trapping and flash injection into the

inlet of a gas chromatograph is described.

An investigation was conducted into the nature and

origin of an unpleasant lachrimatory component emanating

iv

from an industrial furnace, and a number of trapping and

separation techniques were used to determine the nature of

the irritant. The experimental methods used are described,

and conclusions are drawn concerning the suitability of the

various techniques for trace organic volatile sampling.

The steam volatile components of the oil of two

Tasmanian native plants, Drimys lanceolata and Prostanthera

lasianthos are examined using gas chromatography retention

criteria on two columns and mass spectral information. A

comparison of the volatile components of Boronia megastigma

in the flowers, concrete, and.concrete headspace is

described and the application of the technique to the

determination of the optimum time for harvesting, is

outlined.

Changes in the volatile components produced upon injury

of Pinus radiata are examined using headspace traps above

and below the injury and comparisons are made of the

volatile components present in the bark.

ABBREVIATIONS

GC - Gas Chromatograph

FID - Flame Ionisation Detector

MS - Mass Spectrometer

TIC - Total Ion Current

HPLC - High Performance Liquid Chromatography

CW2OM - Carbowax 20M Stationary Phase

OV101 - OV Silicone 101 Stationary Phase

GLT - Glass Lined Stainless Steel Tubing

SCOT - Support Coated Open Tubular (Column)

WCOT - Wall Coated Open Tubular (Column)

MIKES - Mass-analysed Ion Kinetic Energy Spectrometer

PMD - Programmed Multiple Development (Chromatography)

TDL - Toxic Dose Level

ppb - Part per billion

ppm - Part per million

Deg.C-Degrees Celsius

1

INTRODUCTION

The identification of components in complex mixtures of

volatile organic substances, plant volatiles, working and

living atmospheres, and volatile substances of forensic

interest, has been limited by the techniques available for

collection and identification.

Identification of compounds in some workday situations

has established the presence of a number of carcinogenic,

mutagenic and teratogenic agents as well as those known to

cause liver and other vital organ malfunctions. An

increasing awareness of the influence of some volatile

components on human health has created the need for a

generally acceptable group of techniques for their

separation and identification.

Major problems associated with the investigation of

volatile components include the degree of uncertainty of

identification due to formation of rearrangement products

and artefacts, and to laboratory contamination. Volatile

organic materials present in the atmosphere of work places,

industrial areas, hospital operating theatres, city streets,

waste chemical dumps all present some degree of hazard to

human well-being. This thesis examines the concentration,

separation and identification-of some volatile organic

mixtures present in a number of distinct but related areas.

2

CHAPTER II

EXPERIMENTAL TECHNIQUES FOR THE CONCENTRATION AND ANALYSIS

OF VOLATILE ORGANIC COMPOUNDS.

Introduction

The sampling of volatile organic materials in a variety

of atmospheres ranging from the confined air in a space

capsule [1] to background levels in isolated geographical

regions of the earth's surface requires collection,

concentration and identification techniques with varying

degrees of complexity.

Individual component monitoring and identification is

essential in an enclosed area, such as an industrial site,

where the need to recognise the health hazard that specific

compounds have for persons continually exposed to volatile

organic materials. The tracing, fingerprinting and

identification of odorous volatile components in other areas

such as processing plants, sewage treatment works and

abattoirs would also provide information on the degree of

risk of exposed persons to hazardous compounds. The

qualitative and quantitative trapping and identification of

components would provide some scientific basis for the

measurement of odours and some other parameters affecting

what is loosely termed "the quality of life" in an

increasingly more crowded environment.

3

A. Concentration and Trapping Techniques

Techniques for the sampling of volatile organic mixtures

in the atmosphere necessitate the concentration of

components to a point where separation and measurement is

within the capabilities of the analytical instrumentation.

The concentration of components by factors of one hundred to .

ten thousand are often required.

Trapping or concentration techniques may be considered

under three major headings: condensation methods,

adsorption methods and headspace techniques.

(i) Condensation or Cryogenic Methods

Earlier attempts to concentrate volatile organic

materials from air involved the passage of litre quantities

of air through a stainless steel or glass U-tube immersed in

a freezing mixture of solid carbon dioxide/acetone, or

liquid nitrogen [3 -5]. Volatiles and semi- volatile

components of cigarette smoke [6] and food products [7] have

been trapped utilising this technique. Detection limits of

2 ,ug/m have been claimed [8]. In 1967 Willis [9]

described a trapping system involving the cryogenic

collection of a sample in one loop of a gas chromatograph

(GC) separating column. Improved resolution was claimed

especially for lOw molecular weight (Cl to C5) hydrocarbons.

However, as a rapid routine method of sample trapping there

are obvious practical limitations; for example the carriage

of cryogenic liquids to remote or awkward situations (such

as the inside holds of ships) [8].

Baker in a series of articles [10,11], summarised and

reviewed some earlier theoretical and practical aspects of

trace organic contaminant concentration by freezing. A

major problem associated with cryogenic trapping procedures

is the co-condensation of considerable quantities of water

and the inherent losses of low molecular weight water

soluble compounds upon its removal. Tyson and Carle [12],

circumvented this by passing the cryogenically trapped

water-organic mixture through a preparative GC to separate

the water and then through a gas chromatograph/mass

spectrometer (GC/MS) for identification of components

(Figure 1). High recovery efficiencies were obtained for a

selection of terpenes.

Pa" MPAROME 001051

1412 ir eh ! m 0(110101

511000

FLOW

SECOND S ACE SEW 550,11

VENT Two AA? I r I 1i?001000

UOVTICAL 00550100VENT

0 (:—:11 1110F1 2.51111.1 ,(4 M AUER. FLUE

SUM I 1 qu

TOlassoC4 I 1:11/ATIOI VASS

RAP , f

SW f sw MU 01110100 SPECTOONETEI

NEM

SAMPLE IN

FIAST SEW IRA?

CRYOGENIC PRECONCENTRATOR

PREPARATIVE GAS CHROMATOGRAPH

GC—MS

Figure I. Schematic Diagram of a Preconcentration and Analytical System [12].

Cryogenic trapping in conjunction with hydrophobic

porous polymers is an effective method for sampling low

boiling point organic compounds such as ethylene [15].

Teranishi [13] utilised cryogenic trapping procedures for

collection of volatile metabolites from human breath and

^

5

urine; and Copier [14] trapped eluent from a GC on cooled

glass tubes for subsequent IR analysis.

(ii) Adsorption Methods

(a) Carbon Trapping

Carbon in various forms has been widely used for

collecting organic compounds from air [16,17] and water

[18,19]. Activated carbon has differing adsorption

properties depending upon its source material (coconut,

bone, coal or wood), and its particle size. The use of

activated carbon for the economic removal of trace organic

compounds from water has been of increasing importance

following legislation introduced by the U.S. Environmental

Protection Authority, brought about by the discovery of

carcinogenic compounds in New Orleans water [20]. Volatile

halogenated hydrocarbon concentrations have been limited by

U.S. legislation to 0.5 pg/L.

Small quantities (25mg) of carbon have been used by Grob

and Grob [21] to trap microquantities of organic material

from the air. However, with small absorbent weights, air

flow rates need to be low ca. 2.5 mL/min, and sampling

times, as a result, have to be extended to allow collection

of sufficient weight of sample. A wide range of molecular

weight compounds have been collected and analysed by this

method, despite some comment [22], that catalytic changes

and irreversilbe absorption of higher molecular weight

compounds may occur.

6

Desorption of trapped components from activated charcoal

has been effected by vacuum [23] and steam [24], but the

most efficient method appears to be solvent extraction

[25,21,26], using dichloromethane or carbon disulphide.

Rubenstein [27] covers some of the theoretical aspects

of sorption using activated carbon as an example.

b) Coated Support Trapping

The equilibrium concentration of trace organic compounds

upon a liquid stationary phase has been utilised by a number

of authors [32,33], for collection of volatiles from air,

and other gases, to enable subsequent identification and

quantitation by GC. Quantitative trapping of small

quantities of organic components at ambient temperatures on

a support bonded silicon phases has been described in an

excellent paper by Aue [34]. The elution method described

involved reflux solvent extraction and concentration of

solvent before injection into the GC. Loss of volatile

fractions is likely to occur as a result of this latter step

and this problem is mentioned in the paper. Another

difficulty likely to be encountered with the use of support

bonded coatings and solvent extraction techniques is the

possible removal of very minute quantities of coating and,

the degradation of labile components under warm solvent

reflux conditions. Bellar et al [35] described an earlier

sample trapping system using a combination of liquid phase

coated firebrick and cryogenic condensation to identify more

abundant atmospheric hydrocarbons at concentrations of 0.1

7

ppb in 300mL of air. A system of collecting and

transferring amounts as small as 10mg of substances of

varying volatilities utilising coated supports is discussed

by Bierl et al [35a]. Percentage recoveries varied from

seventy to one hundred. However, only single compounds are

mentioned and the system described would not appear to have

the versatility to trap and recover a wide range of

compounds due to the selectivity of liquid phase on the

packing material.

c) Porous Polymer Trapping

Microporous polyethylene was first used as a low

temperature support in 1963 by Baum [39]. A number of

reviews [40] and papers [41-47], have documented the more

recent developments of this packing and trapping material.

Various types of porous polymers are widely used for

separating water, alcohols and aldehydes [48] ao,s well as for

the analysis of low molecular weight gases [49,50].

Authors differ in explanations as to the mechanism of

adsorption and desorption of porous polymers when used for

trapping and elution of organic compounds. Hollis [40]

suggested that the solubility of the compound in the polymer

is the most important factor in determining the order of

elution, whereas Smith and Waddington [51] indicated that

within a class of compounds there is a linear relationship

between log retention time and boiling point of the eluent.

However, pore size distribution, micropore volume, nature of

the polymer and surface activity are all important features [52].

8

Dave [53] reviewed and summarised the information

available on porous polymers up to 1969. (Table 1).

Porous Polymer Surface Area

m 2/g

Average Pore Dia.

(Angstrom)

Water Affinity

Temperature Limit

(Isothermal) °C

Chemical ,Composit• ion

"Chromosorb" 101 30-40 3500 Hydrophobic 300 STY-DVB



Porapak N 437 - Hydrophobic 200 -

Porapak P - - Hydrophobic 250 DVB-STY

Porapak Q 840 74.8 Hydrophobic 250 EVB-DVB

Porapak Q-S - - Hydrophobic 250

Porapak R 780 75.6 Hydrophobic 250 NVP

Porapak S 670 76.0 Hydrophobic 300 ?

Porapak T 450 91.4 Hydrophobic 200 EGMA

PAR 1 100 200 Hydrophobic 250

PAR 2 300 90 Hydrophobic 250

Tenax GC 19 720 Hydrophobic 375 DPPO

XAD-2 300 90 Moderate 200 DVB

DPPO = 2,6-diphenyl-p-phenylene oxide; STY = styrene; DVB = divinyl benzene; EVB = ethyl vinyl benzene; NVP = N-vinyl pyrrolidone; EGDMA = ethylene glycol dimethacrylate.

Table I. A Comparison of the Physical Properties of Some Porous Polymers [53].

9

The development in 1972 by Applied Science Laboratories

of a new porous polymer, 2,6-diphenyl-p-phenylene oxide

(Tenax GC) provided a versatile, efficient, thermally stable

trapping medium that has been widely used for trapping

volatiles from air [2,54,55,56], water [2,57], and

biological samples [54,58].

A number of authors have compared the various types of

porous polymers available for sample trapping and have

reached different conclusions [59], Butler and Burke [60]

after an investigation utilising test samples of t-butanol,

methyl ethyl ketone, benzene and acetonitrile, concluded

that Porapaks Q and R had the best overall sampling

capacities. However, the statement was made that Tenax GC

may be the absorbent of choice for samples consisting of

high boiling components because of the high temperature

limit.and relatively low retention volumes. These

characteristics allow sample components to be desorbed more

rapidly from Tenax than from other adsorbents.

Zlatkis et al have used Tenax GC as a trapping system

for volatiles from human breath [54], urine [54,61], and

plasma [58], but do not appear to have critically evaluated

the method for loss of volatile components. Pellizzari et al

have conducted the most systematic comparison of collection

efficiencies [62], and field sampling variables with Tenax

GC [63]. The effects of humidity, background load levels,

repeated re-use of sorbent, and transportation and storage

of collected samples were investigated. Tenix GC was

- 10-

superior to other sorbents in most cases, percentage

recovery by thermal elution procedures being better than 90%

at the 50 and 100 mg level [64].

Another porous polymer, XAD-2, has been widely evaluated

[65] and used [66-68], for the trapping of trace amounts of

organic materials mainly in water. Claims are made that the

concentration range extends from 5 parts per trillion (10 11 )

to 50 ppm [65]. It should be noted that some of the claims

made in this paper especially concerning recoveries of some

higher molecular weight acids have not been verified in this

laboratory [69]; Nevertheless, the method has found many

applications in trace organic water analysis [70, 71]

despite some of the limitations posed by the removal of

water from the organic extract and the losses entailed.

Morrison et al [70] claimed that "thermal desorption

makes quantitative analysis difficult". This view contrasts

with the opinion of a number of other authors [72 - 74], who

have successfully used Tenax GC and thermal desorption

techniques. As a result of the improvement in sample

collection efficiency and the coupling of powerful capillary

GC mass spectrometer computer systems, over one hundred

volatile components have been collected and identified from

atmospheric sources [56] using Tenax GC. Ciccioli et at

[75] compared the recoveries of Tenax GC and concluded that

a mixture of 0.25g of Tenax GC and 0.45g of Carbopac B

provided a good compromise for the trapping of most volatile

organic compounds from open air and industrial sites. (Table II)

A cryogenic trapping procedure however, was advocated

for low boiling point compounds up to the boiling point of

pentane.

Dressler has published a review of applications of

porous polymers for the extraction of trace organics from

aqueous samples [76] and commented on aspects of method

sensitivity and sample storage.

- 12 -

Compound ' tarbopack B Tenax GC

0.5 L sample

1.5 L sample

5.0 L sample

0.5 L sample

1.5 L sample

5.0 L sample

Methanol 3 0 0 1 0 0

Ethanol' 3 0 0 1 0 0

Methyl chloride 1 0 0 3 1 0

Acetone 5 1 0 68 2 0

Chloroform 100 54 1 100 84 5

Diethylamine 100 80 50 80 50 r

Isobutanol 100 100 25 100 95 16

n-Pentane _ 100 100 100 100 50 9

Cyclohexane 100 100 100 100 50 9

n-Hexane _ 100 100 100 100 100 20

Ethyl acetate 100 100 100 100 100

n-Butanol _ 100 100 100 100 100 35

Benzene 100 100 100 100 100 35

Toluene 100 100 100 100 100 100

* _ - - - - - -

ra-C 13 65 65 65 100 100 100

n-C14 46 46 46 100 100 100

11-C 15 25 25 25 100 100 100

11-C16

8 8 8 100 100 100

.11-C17

1 1 1 100 100 100

ri-C18

0 0 0 100 100 100

*Styrene, ethylbenzene, xylene, pyridine, chlorophenol, alkanes, alkenes from C

7 -C12 had 100%.

Table II. Recovery (percent) of Organic Compounds from Air Samples Using Carbopack B and Tenax-GC Traps.

- 13 -

d) Miscellaneous Adsorption Techniques

Activated alumina [77] and molecular sieve 5A [30] have

also been used to trap volatile organic compounds from the

atmosphere. However, both materials tend to irreversibly

adsorb some of the higher molecular weight labile components

and to show the same disadvangages as silica.

(iii) Solvent Trapping

Solvent scrubbing of organic components from air [74]

has met with only limited success. The reduction of solvent

volume in order to achieve the required final concentration

may be accomplished with minimal losses of high boiling

components but significant losses of volatile compounds do

occur.

The design by Goldberg et al [78], of a closed loop

solvent extraction system for trace organic compounds in

water, has enbabled the concentration of components by a

factor of 10,000. The system operates with any water-

immiscible solvent, however, the selectivity of the solvent

for particular compound classes and the high losses

associated with concentration, as mentioned above, tend to

limit the applicability of the method.

Grob [79] refined the solvent extraction procedure and

extracted 1 litre of tap water with 200 microlitres of n-

pentane and evaporated the solvent to 3 microlitres for one

GC injection. (Figure II). Over fifty components were

separated and identified by this method.

- 14 -

Figure II. GC Traces of Pentane Extracts of Water Samples [79].

Column: 2 m x 1/8" steel column, with 4 % Carbowax 20 M on Chromosorb G. AW-DMCS, 80-100 mesh

Column temperature: 65 °C Sampling capillary: 150 °C Carrier gas: Argon 28 ml/min Scavenger gas: 100 ml/min,

95 % argon. 5 % methane Detector: ECD Ni63, 1 nA Attenuation: x 64 Sample temperature: 90 `C Sample: 1 ml pale ale.

injection time 8 sec Components:

1 = diacetyl (approx. 0.08 mg/I)

2 = pentan-2.3-dione

fir

Head Space Analysis of Beer The head space technique has also proved to be a valuable tool for beer analysis. The two chromato-grams shown below demonstrate practical application in routine operation. The left-hand figure (A) gives the detection of the higher alcohols and esters, and the right-hand figure (3) shows the selective detection of vicinal diketones with art ECD.

Column: 2 m x 1/8" steel column, with 15 % Carbowax 1 500 on kieselguhr

Column temperature: 60 °C • Detector: FID Attenuation: x 2 Sample temperature: 40 'C

Use Of the Head Space Technique for Environmental Analysis Detection of hydrocarbons in waste water Column: 2 m x 1/8" column, packed

with 15 % polyethylene glycol on kieselguhr

Column temperature: 60 'C Detector: FID Attenuation: x 4 Sample: 10 ml water Sample temperature: 75 'C B = benzene 0.2 ppm T = toluene 0.5 ppm o-X = onhoxylene 0.1 ppm

- 15 -

B. Direct Headspace Collection.

Headspace collection and analysis of volatile organic

compounds is applicable if the concentration of the

components is sufficient for direct detection by the

analysing system. Hence, for GC analysis, one of the

previously mentioned concentration steps is often employed.

Headspace sample collection is a simple, effective method of •

analysing volatile components in a closed vessel. A number

of automatic and semi-automatic systems for syringe

headspace GC analysis are now available commercially [80]

following earlier work by Kolb, Novak and others [81-82].

Figure III illustrates applications of the technique to a

number of different sample types.

30 A 2'0 1 .0 0 mon 15

Figure III. Headspace Analysis of Volatile Components [80].

The headspace sample is drawn from a closed vessel by

means of a gas tight syringe needle through a silicon rubber

or PTFE seal [83,84]. Careful temperature and gas flow

control is required in order to obtain reprodilcable results.

- 16-

Problems with adsorption onto the septum may be encountered

with some volatiles and solvents [85-87]. This effect may

be minimised by use of the more expensive but inert PTFE

seals.

Injection of total headspace volatiles was undertaken by

Loper [88] utilising a heated gas tight syringe in his

examination of alfalfa flowers. Many other applications of

routine headspace analysis are being developed. The chief

advantage being speed in a quality control situation where

samples such as blood alcohol, plastic volatiles, obnoxious

odours, plant ripening volatiles [80,89] are to be

monitored.

- 17-

C. Separation and Analytical Techniques.

The introduction of capillary GC separation techniques

and mass spectrometer-computer data systems, has enabled the

separation and identification of complex mixtures of

volatile organic compounds that would previously have been

very difficult. Examples include the separation of

automobile exhaust and miscellaneous background volatiles

from ambient air [2,75], and human expired air volatiles

[73].

Development work is continuing on the coupling of liquid

chromatographic systems to the mass spectrometer to extend

the variety of sample types capable of being introduced to

this sensitive identification equipment [90].

i) Separation Techniques

Efficient separation of closely related chemical

compounds in complex mixtures is a necessary prerequisite to

positive identification. Unfortunately it is not always

possible to achieve this objective. In many instances

therefore, only a probability of component identification

can be assigned.

a) Gas Chromatography

Capillary GC columns, to date, have provided the most

effective system for the separation of complex mixtures of

volatile organic compounds [91,92]. The characteristics of

small sample requirement, high resolution, lower temperature

and speed make the use of capillary GC columns especially

- 18-

useful. Opposed to this, glass capillary columns are

relatively fragile, require special techniques for

preparation that are normally outside the scope of the

average laboratory, and require somewhat complicated

plumbing arrangements within the GC. Glass capillary

columns are usually drawn from borosilicate or soft glass

that contain levels of metal oxides that may catalyse

stationary phase decomposition at elevated temperatures,

resulting in column bleed.

The use of vitreous silica instead of glass as a column

material provides the efficiency and resolution

characteristics of glass columns while offering improved

inertness, thermal stability, flexibility and mechanical

strength [93,94]. The flexibility allows the positioning of

the end of the GC column through the GC-mass spectrometer

interface to a point adjacent to the ion source [95] thus

reducing the peak broadening effects associated with normal

interface systems. Silica capillary columns are being used

in diverse areas of medical [96], environmental [97] and

flavour analysis [98] to provide resolution of complex

mixtures of volatile components. The wide acceptance of

silica columns is shown by the number of papers presented on

this subject in the 1982 "Pittsburg" Conference held in the

U.S.A. [99]. (Five papers from a total of twelve in the

subject of Gas Chromatography).

The sharpness of peaks in capillary chromatograms often

allows measurement of quantities of materials which would

escape detection when eluted as broad bands of equal area

- 19-

from packed columns [100].

Temperature programming is usually employed due to the

wide range of boiling points represented in complex

mixtUres. Sub-ambient temperature programming has been used

by a number of authors [101] for separation of low boiling

compounds. The advantages of capacity of packed columns and

the efficiency of capillary columns has been utilised by

Blass et al [102] in a versatile combined configuration that

allows repetitive trapping and a number of other operations

within the one GC oven. Figure IV illustrates the

connections required to perform packed column operation,

capillary column operation, or combinations of the two. A

relatively large quantity of a mixture may be injected onto

the packed column and some components held by the trap TD

for further separation on the capillary column.

Figure IV. Two Column GC System - Packed and Capillary with Valve Switching [102].

PPC - Packed Column. CC - Capillary Column. D 1/2 - Detectors. I - Injectors.

Switching Valves, Needle Valves, and Filters.

- 20 -

Development of sampling techniques for trace volatile

organics from atmospheric sources [54,72,103] complements

the recent advances in capillary gas chromatography. Due to

the very fine detail discernible in complex mixtures,

capillary columns find a number of applications in

'fingerprinting' or source tracing in forensic science and

other fields. Examples include identification of oil spills '

[104], air pollution sources [105] and the origin of

Cannabis plant material [106].

Capillary columns may be considered under two headings -

SCOT columns are Support Coated Open Tubular columns with an

internal diameter of 0.5mm, where fine solid particles of

diatomaceous earth support, coated with a film of stationary

phase, are distributed evenly over the wall of the column.

They are suited to the analysis of volatile samples

[107,108] and are capable of accepting a greater sample load

than WCOT columns. Wall Coated Open Tubular columns have an

internal diameter of approximately 0.25mm and are used where

maximum resolution is required. A thin film of liquid phase

of 0.2 to 0.7 micron thickness is deposited on the inner

surface of the column. The inner wall is etched to provide

the correct surface properties and good mechanical support

for the liquid phase. Due to the thin film of liquid phase

the loading of this column type is restricted in comparison

to SCOT and packed columns. An inlet splitter is frequently

used to deposit the optimum quantity of material onto the

column - the remainder of the sample or solvent being

vented.

- 21 -

b) Mass Spectrometry (MIKES)

The mass spectrometer may be used as an efficient

separation/ identification tool when coupled to data

reduction and library search computer facilities. The direct

introduction of a complex mixture of volatile components

into a MS, the use of computer controlled scanning and

selected ion monitoring, has enabled the separation and

identification of components [109], and the study of

reactions and reaction mixtures [110,111]. The method is

based on the mass-analysed ion kinetic energy spectrometer

(MIKES) first described by Beynon et al in 1973 [112].

Sample preparation is minimal - in some cases none at all -

and the technique is useful for structural elucidation.

However, quantitation has been difficult to achieve [113]

and further developments are required to make the technique

amenable to routine work.

c) High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) has not

been widely used An the past as a separating method for

volatile organic mixtures. However, recent advances in HPLC

technology have made the technique comparable to GC in

speed, convenience and efficiency [114,115]. The chief

advantage of HPLC is the fact that milder solventrand thermal conditions

may be employed for the separation of labile components that

may decompose under harsher GC temperatures. Precursers of

many volatile components are labile and HPLC .could prove to

be of value in determining breakdown pathways -. One of the

- 22 -

disadvantages of HPLC has been that the separation

efficiency of HPLC columns (approximately) 7,000 theoretical

plates) has been a lot lower than the 120,000 theoretical

plates of silica capillary GC columns. Recycling of the

column effluent and the use of microbore columns can improve

the resolution [116]. However, peak capacity is reduced and

complex mixtures with a wide range of boiling points cannot .

be effectively separated by this procedure.

HPLC has been widely used for the separation of polynuclear

aromatic compounds in airborne particulate material [117],

and to complement and extend the determination of this class

of compounds in conjunction with GC techniques [118].

d) Miscellaneous Separation Techniques

Gel permeation chromatography [119] has been used for

separation and molecular weight determination of labile

substance of similar molecular size [120]. Hendrickson

[121] referred to the method as "a new basic tool that could

be called a liquid phase size spectrometer". However, this

chromatography technique is applicable to compounds of high

molecular weight and low volatility.

Liquid-liquid extraction systems are now available

commercially as countercurrent chromatography apparatus.

Claims are made [127] that the method combines the benefits

of countercurrent distribution apparatus and liquid

chromatography, with minimium sample loss, no contamination

from solid supports and no tailing of solute peaks due to

- 23 -

adsorption effects. Other liquid chromatography techniques

such as droplet countercurrent chromatography [122] have

separation efficiencies close to that of GC, but also have

limited applicability in trace volatile separations except

where, as above, labile components are encountered.

Liquid-liquid fractionation techniques are frequently

used as preliminary separation steps where complex mixtures

of organic compounds are separated into various classes

[123-125]. The combination of chemical information obtained

by fractionation, GC and/or HPLC retention data coupled with

spectroscopic information is usually sufficient to identify

components of complex mixtures with reasonable certainty

[126]. The disadvantages of fractionation methods of sample

separation are twofold, involving the efficiency of

separation under slightly differing conditions, and problems

concerning the formation of artifacts or the introduction of

impurities. Many authours fail to provide data on these

aspects of the technique [126].

Programmed multiple development chromatography (PMD) is

a method of processing thin layer chromatography

plates[128-132] to improve resolution and sensitivity. A

system of sequential solvent advance and evaporation ensures

reconcentration of spot material along the axis of 'solvent

motion, producing a thin band rather than a diffuse spot.

Advantages claimed are:- shorter development times, improved

resolution of up to 50 times compared with conventional TLC,

sensitivity improvement of 5 to 10,and a greater tolerance

-24 -

to overloading. Most of the above techniques have only

limited applicability to volatile trace organic separation

systems.

- 25-

D. Detection and Analytical Techniques.

Trace volatile components eluted from a GC are usually

identified according to the retention time compared to a

standard under identical conditions. Incorrect

identification may result if several compounds elute at the

same time, or if a massive amount of solvent alters

conditions in the column.

• The flame ionisation detector combines sensitivity and

versatility for trace compound detection. However a range

of on-line spectroscopic detectors is available [133]; and

the selectivity of the electron capture [134] and flame

photometric [135] detector is widely utilised. Development

of a helium microwave emission detector [136-138] has

enabled the determination of trihalomethanes in 10 ml of

drinking water to levels of 1 part per billion [139].

The most powerful combination for separation, structural

determination and quantitation is thecapillary GC/MS -

computer system [21], [91 - 92], [140-144]. This combination

is capable of the separation and identification of complex

mixtures of volatile organic materials in a variety of

matrices [145-146]. The data processing capability allows

the storage, retrieval and tentative identification of

several hundred component peaks of a complex mixture

[147-148], [149-150].

Data processing systems coupled. to GC/MS can.be used to

resolve overlapping GC peaks and to counteract unwanted

. SO

Scan Number

rnis

Individual " t Mass -251 Chromatograms Reconstituted

Mass Spectra 4.5

1St

ORIGINAL GAS CHROMATOGRAM

LS

Mass Reconstituted Gas Chromatogram

Scan Number

• 7.

- 26 -

contributions from column and septum bleed that would

otherwise distort the relative intensities of ions in the

mass spectrum [151-153]. The resolving effect relies upon

the distinction between the MS parent ion peaks, which may

be detected even though the original GC peaks were over-

lapping (see Figure V). Usually 5-10 mass spectra can be

collected per GC peak and this provides enough information

for processing and reconstruction of a resolved mass

chromatogram.

Figure V. Complex Peak Resolution by MS-Computer Techniques.

- 27 -

Sweeley and co-workers have developed methods for

separating complex mixtures using computer programs to

control repetitive scanning and selected ion monitoring

[154]. They have developed a system that incorporates the

use of retention indices with off-line reverse library

searching of selected mass chromatograms from repetitive

scanning of the GC/MS of complex mixtures. Observed

precision of retention index was 0.2 percent and lower limit

of detection lOng. The GC/MS precision was 8 percent within

duplicate detection of the same sample and the linear range

for quantitative ananlyses was 1,000 fold [155].

The most significant, advance in techniques in this field

have been the combined utilisation of the developments of

splitless sample injection, the preparation [156], and

evaluation [157] of the operation and performance of WCOT

columns, open split injection [158] and direct connection of

the GC capillary to the ion source of the mass spectrometer.

Other detection and analysis techniques for monitoring

volatile components include; Fourier transform spectroscopy

[159-161] using folded path optics for the measurement of 10

ppb levels of hydrocarbon and other species; infrared

spectrometry coupled with microprocessor control [162],

chemiluminescence [163] as well as a variety of laser based

systems [164-165].

Vapour phase infrared spectrophotometric.detection of GC

eluant peaks [166] using on line computer techniques has

- 28 -

recently shown to be effective in some applications where

mass spectral techniques are inadequate [167].

Ancillary techniques, or modified computer search

methods, need to be utilised if positive identification of

some components is required. Comparison of published mass

spectra of terpenes [168], for example, indicate that

structurally different terpenes such as tricyclene, alpha-

pinene, and 3-carene show differences only in ion .

intensities of the principle ion peaks with identical mass.

These differences are often within the normal operational

variables if there are other contributing factors such as GC

column bleed or incomplete peak resolution and hence,

unequivocal identification by mass spectral means alone is

not possible [169].

So rapid are the changes in GC/MS - data system

applications that the quantity of literature published in

the field of trace organic analysis using this combination

of techniques is extremely difficult to scan. The various

reviews in Analytical Chemistry provide a useful method of

gaining some overview of the topic. A summary of detection

systems for trace organic volatiles may be found in the

Application Reviews of Analytical Chemistry [170] and also

under the specific headings of Mass Spectrometry [171] and

Gas Chromatography [172] in the Fundamental Reviews.

- 29 -

E. Sources of Error in Trace Volatile Analysis.

Many organic compounds present in air and water are

labile and the method of sampling will influence the

qualitative and quantitative result of any analytical

programme. The use of complementary techniques for sampling

and analysis and the choice of conditions that tend to keep •

decomposition and rearrangements to a minimum should be

utilised wherever possible. Contamination of a sample at

any stage of the collection analysis chain may lead to false

assumptions and costly errors. A well documented example

was the contamination of lunar rock samples [173-175] and

the difficulty in establishing if the substance measured was

a contaminant or otherwise.

An accurate measurement has been defined [176] as a

precise numerical value that is free of, or corrected for,

all systematic errors. In order to attain such a result,

all aspects of sample collection, storage, extraction,

concentration, isolation, identification, and quantitation

must be examined. Lewis [177] has discussed some of these

problems with special emphasis on trace organic analysis.

The correct choice of sampling method to ensure a

respresentative sample depends on factors such as

representative volumes, times and avoidance of contamination

by the sample collection and analysis method. Each

succeeding step should be undertaken in the shortest

practical time, however if storage is necessary a

- 30 -

temperature of 0 deg.0 or less should be chosen to minimise

effects of photo- decomposition, microbiological action, and

chemical interaction between components and container [178].

During the extraction of volatile components from air

and water, contamination and losses may occur from a number

of sources; solvents and solid extraction systems contain

impurities, and extraction efficiencies are often

considerably less than and extraction efficiencies are often

100 percent, especially where a wide range of compound types

are involved. If a solvent extraction procedure is employed

the components in the solvent may require concentration

prior to analysis. Loss of volatile components and reaction

of labile compounds often occurs as a result of this step.

The use of Kuderna-Danish concentrator [179] can help to

minimise the loss of volatiles provided care is taken in

heating and the choice of solvent. Concentration to

approximately 50 microlitres is possible with a micro-

Kuderna-Danish concentrator. XAD and Tenax resins are also

used to concentrate components as outlined previously,

however volatile losses may also be significant using this

technique and should be monitored where possible.

Problems with isolation of individual components

involving rearrangements, irreversible adsorption,

decomposition and incomplete separation are common to

chromatographic and MS methods. Optimisation of

instrumental parameters and correct choice of technique for

the sample type will help to minimise many of the effects

mentioned.

-31 -

Identification of a component by comparison with

properties of a previously measured known compound may be

extremely difficult where complex mixtures of trace organic

materials are to be analysed e.g. drug, food and plant and

pesticide breakdown products. Pure standards for these

compounds are often not available to allow addition

techniques to be employed for positive identification.

Combined GC/MS systems employing internal standards

incorporating carbon 14 have been extensively utilised

[180] as an internal quality control system. Addition of an

isotope labelled labelled compound is an effective method

for the determination of losses of components during

subsequent extraction, concentration and isolation steps.

Isotope labelling cannot, however, be undertaken for each

component in a complex mixture and differences in chemical

nature between compounds can cause variations in recoveries.

One compound of each class likely to be present should be

selected to check on recoveries through the system.

A variety of methods have been employed to assess

accuracy in trace organic analysis [178]. These include

collaborative laboratory studies, analysis of spiked samples

and inter-laboratory comparisons [181]. Many of these

studies have shown the need for more extensive in-laboratory

quality control checks, and the necessity for the

development of a wide range of appropriate Standard

Reference Materials [182].

-32 -

CHAPTER III

,DEVELOPMENT OF A SAMPLING SYSTEM FOR VOLATILE

ORGANIC MIXTURES.

Introduction.

A simple method of sampling volatile compounds from a

variety of sources is required in many applied areas of

chemistry. The detection and analysis for example, of

volatile solvents in paint spraying industries, odour

tracing in environmental problem areas, and plant volatiles

for the perfume and flavour indistries are some typical

examples where the use of a rapid routine method of

collection and analysis of volatile compounds would be of

advantage.

Ideally a sampling system should be capable of trapping

quantitatively and reproducably a wide range of chemical

compounds in sufficient quantity to give an accurate

analytical assessment. The trapping method should not

degrade or alter the collected components on collection,

storage or elution. The above properties of a trapping

system are often difficult to effectively check,

particularly in the case of complex mixture trapping. Upon

• adsorption, there is a possibility of chemical reaction

between the components trapped, due to the higher

concentration in the medium, with other species and also

with the trapping medium.

- 33 -

investigation of the available information in the literature

was made in order to select the adsorbants with the most

favourable characteristics. An outline of this literature

search is provided in the previous chapter. A number of

different sample collection and elution methods were

selected, tried and some rejected, as described in the

following pages.

-34 -

A. Selection of a Sample Adsorption Medium.

(i) Carbon,

The efficiency of carbon as a trapping material in the

form of 25mg charcoal disks, as demonstrated by Grob [21],

prompted the investigation of this material as a trapping

medium for the collection of organics from ambient and

contaminated air. Difficulties encountered involved the

manufacture of a consistant porous charcoal filter that was

mechanically rigid enough to withstand the force of the air

passing through the structure. The original commercial disks

as described by Grob were not readily available. Various

modifications of a dye press were tried and varying amounts

of heat were applied to the carbon in order to obtain a

consistant useable product - without a great deal of

success.

Crushed granular carbon (activated charcoal) from B.D.H.

Ltd. U.K., "Special for Gas Adsorption", of two screen sizes

was cleaned by Soxhlet extraction with methanol and

dichloromethane as solvents. Each size fraction was packed

into 9mm diameter by 95mm long glass tubes with a 310 cone

at one end and held in position by glass wool plugs at the

ends. Material similar to this is extensively utilised for

removing organics from air in gas masks[190].

Recovery checks on a range of compounds yielded the

information in Table III. The test compounds were

introduced into a flowing nitrogen carrier gas stream by

- 35 -

controlled addition using a Waters HPLC pump feeding into a

mixing vessel. The liquid sample flow rate in conjunction

with the gas flow rate was adjusted to give a concentration

of 100 p m (Figure VI).

1 hi

Figure VI. Flow Diagram of Recovery Test Apparatus.

1. HPLC pump. 2. Thermostated mixing vessel. 3. Carrier gas inlet valve and flowmeter. 4. Bleed valve and flowmeter. 5. Sample trap. 6. F.I. detector.

Size Fraction -10+20 Mesh -200+250 Mesh

Pentane 95 98 Hexane 96 96 Acetone 85 92 Methanol 92 93 Ethanol 83 87 Benzene 82 86 Ethyl acetate 78 68 Carbon tetrachloride 83 86 Diethylamine 64 58

Table III. Percentage Recovery Test Activated Charcoal Adsorbent. Test Compounds at a Concentration of 100ppm.

The flame ionisation detector of the GC was used to

determine the breakthrough of the test compound and the

sample tube was then removed for solvent extraction and

recovery. A second recovery check was made by adding one

microliter of the steam distillate of Pinus radiata to the

top of the column and washing in with 500 microliters of

- 36 -

carbon disulphide. Extraction from the activated charcoal

was accomplished by refluxing 5mL of carbon disulphide

using a cold finger apparatus as shown in Figure VI. The

small volume refluxing through the trap reduced the

concentration of the impurities upon solvent reduction in

the micro Snyder distillation (Figure VII). An internal

standard of n-decane was added to the carbon disulphide at

the completion of the extraction to enable allowances for

some of the losses likely to take place during the solvent

reduction step in the concentration apparatus.

Recovery of the lower molecular weight components of the

test compounds was quite acceptable but losses of higher

molecular weight components, especially oxygenated terpenes

of the radiata pine oil, was unacceptable. For this reason

the use of activated charcoal was not continued.

2.

Figure VIIa. Micro Reflux Apparatus.

1. Cold Finger. 2. Sample Tube. 3. Solvent Flask (10mL).

. 2.

Figure VIIb. Snyder Concentration Apparatus.

-37 -

1. Snyder Distillation Column. 2. Heat Shield Covered With Al

Foil. 3. Graduated and Calibrated Taper. 4. Hot Plate.

- 38 -

ii) Tenax GC

Tenax-GC has been widely utilised for the concentration

of trace organic compounds in air as described in the

previous chapter. Tenax (poly-p-2,4-diphenylphenylenoxide)

has the structure shown in Figure VIII and the manufacturers

claim it is stable up to temperatures of 350 deg.C.

Figure VIII. Chemical Structure of Tenax GC.

The thermal stability and hydrophobic nature of the

material coupled with the ability to trap a wide range of

organic compounds would appear to make Tenax a favourable

choice as an adsorbant.

Tenax was prepared by conditioning overnight at 325

deg.0 in a wide bore glass tube in a stream of high purity

nitrogen. The packing was stored in capped glass containers

prior to packing in sample tubes and then reconditioned at

275 deg.0 before use.

Recovery Checks. Pellizzari et al [63], have recorded

the breakthrough volumes of a number of volatile organic

compounds. Checks were made on many of the figures

presented using a 4mm internal diameter by 170mm long glass

tube packed with Tenax (Table IV). The results shown were

- 39 -

calculated back to 2.15g of Tenax for comparison [187].

There was substantial agreement for all compounds except

acrolein. Apparatus used for the test was similar to that

of Figure V.

Breakthrough Volume (L)* Compound 2.159 Tenax 2.87g Tenax Lab.

Comparison

Acrolein 9 13 3 Propylene oxide 15 20 15 Diethyl sulfate 19 25 18 Trichloroethylene 50 - 52 Nitromethane 86 114 87 Glycidaldehyde 147 195 - Chlorobenzene 300 - 310 B-propiolactone 330 440 Bis-(chloromethyl)ether 400 530 Butadiene diepoxide 646 860 Cyclohexene oxide 1,040 1,380 1,100 N-nitrosodiethylamine 1,220 1,620 1,200 Aniline 2,100 2,800 2,250 Ethyl methanesulfonate 2,420 3,220 - Styrene oxide 7,550 10,000 7,600 Acetophenone 2,880 3,380 2,850

*Volume required to elute one-half of adsorbed vapour at 25 deg.C.

Table IV. Breakthrough Volumes for Several Highly Volatile Compounds on Tenax GC (40/60 Mesh) Cartridges

Janak [188] and other authors, have confirmed the

negligible influence of water vapour on the trapping and

elution efficiency of Tenax. Tests conducted in this

laboratory at 50% and 70% humidity levels, using steam

distillate of Pinus radiata bark oil, showed no alteration

of trapping and recovery efficiency.

- 40 -

B. Development of Sample Collection and Desorption Equipment.

i) Sample Collection

Optimum sample collection efficiency is dependent on

factors such as gas flow rate, rate of diffusion of sorbate

into the pores, particle size and shape, bed geometry,

porosity and temperature [187]. The geometry of the sample

collection tube was limited by mechanical aspects of the

desorption apparatus as well as theoretical considerations.

The equipment used to collect volatile organics from air

samples consisted of:- 1. a Pyrex glass or metal tube packed

centrally with 40-60 mesh Tenax held in position with

silanised glass wool. The weight of Tenax per sample tube

was 0.40g. 2. A calibrated flowmeter measured the rate of

gas passing through the sample tube. 3. An electrically

driven diaphragm pump was used to draw the gas through the

system (Figure IX).

• . •

3 ..

Figure IX. Flow Diagram of Apparatus for Sampling of Volatile Organic Compounds.

1. Glass Tenax trap. 2. Calibrated Flowmeter. 3. Diaphragm pump.

Flow rate for sample collection was 30-50 mL per minute

and the temperature of the sample tube was maintained at or

below 20 deg.0 (by a water cooled jacket if required).

- 41 -

ii) Sample Desorption

The requirements for thermal desorption and injection of

the sample into a GC include quantitative removal of the

sample from the collection medium, and rapid injection of

the vapour into the inlet of the GC without bleed during

secondary trapping.

A trapping system described by Houghton [189] and

illustrated in Figure X, was constructed with some

modifications. A luer adapter was silver soldered onto a

6mm Swagelock fitting to allow the connection of a syringe

needle to the metal sample tube using a compression Graphlok

seal. The equipment consists of a heating coil mounted on

two metal formers with a gap between to allow the insertion

of hot or cold metal probes [190]._

A

El Glass Wool Coated Packing

ari Asbestos Jacket Yi.j: Asbestos Tape

• Nichrome Wire

-7-Figure X. Initial Thermal 'Desorption Apparatus [190]. (A) .Stainless-steel collection tube; (B) cross section

of the collection-tube heater with tube in position.

The sample tube was partially inserted into the

apparatus and a syringe needle attached and inserted into

- 42 -

the septum of the GC. The carrier gas line was attached by

a second Swagelok fitting to the other end of the sample

trap. Heat was applied to the tube by the passage of an

electric current through the heater windings and the

application of a liquid nitrogen cooled trap prevented the

passage of the components into the GC. When the system

reached a steady temperature of 200 deg.0 the cold probe was •

removed and a hot probe placed in the same position. The

carrier gas from the GC at the same time being diverted

through the sample tube to flush the components into the GC.

The system was found to function efficiently for a

single component in air samples and was very good for

isolating single peaks from a GC run. (This was

accomplished using a heated column switching valve to divert

the carrier gas flow from the FID to the trap at the

expected elution time of the peak. A component collected in

this manner could be reinjected into the GC onto a different

column to check for purity:) Problems were encountered

however, with complex mixtures of components with similar

boiling points giving rise to overlapping peaks and poor

recoveries (Figure XI). The trace is of a sample taken from

an organic teaching store exhaust air stream.

FID

Resp

onse

•

20 0 4 8

200 12 16

250 Temp. °C 20 24 Time (mins)

-43-

Figure XI. GC Trace of Trapped and Injected Volatile Organic Compounds in Air (2m OV101 GC Column, 120-260 deg. C at 5 deg./min. Air Sampled 1.96L 20 deg. C )

The problem with the technique appeared to be the

inefficient transfer of heat to the sample tube resulting in

lack of resolution on the analytical column. A variety of

columns, including capillary columns, were used but with no

major improvement in resolving power.

Redesign of the system to give better heat transfer and

improved cold trapping appeared to be necessary. The heating

block was constructed to enable continuous desorption of the

organic compounds as heat was applied, and flushing of the

components by the carrier gas stream to a cold trap

directly immersed in liquid nitrogen.

- 44 -

"2-

Figure XII. Thermal Desorption and Injection Apparatus.

1. 2mm Swagelock Fitting - carrier gas inlet. 2. Tenax Sample Tube. 3. Resistance Wire Round S. Steel Heating Block. 4. 6mm Swagelock Coupling and Electrical Connector. 5. 3m GLT. 6. Electrical Connector and Coupling to GC inlet. 7. GC Column.

A diagram of the equipment is shown in Figure XII.

The apparatus consists of a 25mm diameter stainless steel

cylinder (2), drilled through the centre to accept a 170mm

by 6mm sample collection tube, and off-center to accept a

thermocouple probe. The metal cylinder was covered with a

heating element consisting of 7.2m of 1.779 ohm/m in a

single layer of No. 20 gauge B. and S. resistance wire wound

over mica. Carrier gas tubing was connected to the 2mm

coupling at one end of the heater block (1), and the Tenax

sample tube was inserted intO the other end of the block

through a drilled out 6mm Swagelock fitting silver soldered

- 45 -

to the stainless steel. Gas sealing by a Graphlok ferrule

(4), also prevented the tube from being ejected as did the

coupling of the 3mm diamter glass lined metal U tube (5).

At each end of the U tube brass lugs were attached to

provide electrical connections for coupling to a welding

transformer. A number of different configurations of glass

lined metal U tube were manufactured, the use depending upon

the configuration of the inlet system of the GC or GC/MS

unit. A horizontal version with a luer fitting and a 20

guage needle was frequently used. The system illustrated

shows the 3m glass lined tubing (GLT) coupled directly to a

piece of 6mm GLT inserted into the inlet heater of the

GC/MS.

Cooling of the U tube involved placement of a small

insulated vessel filled with liquid nitrogen under the U

section of the tube. The apparatus was then coupled to the

carrier gas flow and the system purged of air for a few

minutes at a low flow rate. Optimum carrier gas flow

adjustments could be undertaken when the system was

connected to the GC inlet and then heating of the block

could commence. A power. supply of 50V from a variable

voltage transformer enabled the heating block to reach a

temperature of 200 deg.0 within 6 minutes. A chromel-alumel

thermocouple inserted into the stainless steel block was

used to monitor the desorption temperature. Thermal elution

times of 6 to 10 minutes proved to be adequate for most of

the volatile air samples trapped using Tenax: The carrier

gas transported the desorbed organics to the U tube section

-46 -

of GLT where secondary trapping (condensation) concentrated

the material.

Rapid injection of the organic mixture onto the GC

column involved resistance heating of the 3mm GLT by

connecting the secondary output from a welding transformer

across the two lugs, at points 5 and 6, immediately after

the removal of the liquid nitrogen bath. A current setting

of 40A applied for 30 seconds rapidly heated the GLT to a

maximum temperature of 300 deg.C, at a rate that ensured

breakage of the tubing did not occur.

Following a number of field trials, where mechanical

breakage of glass sample trap tubes had occured, the

decision was made to replace the glass with glass lined

stainless steel tubing of the same dimensions. The added

mechanical strength of the GLT also was an advantage in the

desorption apparatus where coupling of the 3mm U tube to the

desorption system had often placed an unacceptable degree of

strain on the glass tube. Heat transfer to the packing from

the heating block was also improved.

The redesign of the thermal desorption equipment, as

outlined above, improved the quality of the traces obtained

from the GC and GC/MS runs and enabled use of the method for

routine sampling of volatile mixtures in a number of areas

that would not have been possible using previously available

techniques.

-47 -

C. Blank Determinations and Sample Trap Storage. •

Introduction.

An important aspect of the sampling of trace volatile

organic compounds is the requirement to reduce the risk of

contamination of the collected sample. Contamination may

occur in a number of ways; from degradation of the

collecting material, from breakdown of the collected

components so that there is no resemblance to the original

sample; or, the ingress of chemicals, such as solvent

- vapours, from the handling or laboratory.

A number of instances occurred, in the course of the

investigations that are documented in the following pages,

where solvents such as chloroform and 'Freon' were detected

in collected air samples that should have been free of such

components. The steps that were followed to reduce the

contamination that tended to result from working in a

general purpose organic laboratory are described.

Experimental.

Tests were conducted on the desorption efficiency at 250

deg.0 and 300 deg.0 on portions of Tenax that had been

previously used fbr trapping terpene volatiles. One tube

was initially conditioned at 250 deg.0 for 60 minutes in a

stream of high purity nitrogen, and the other was

conditioned at 300 deg.0 under similar conditions. Each

sample tube was then placed in the stainless steel

desorption block at 250 deg.0 and 300 deg.0 respectively for

- 48 -

ten minutes and the eluted 'blank' run through the GC/MS

system. An OV101 WCOT 50m silica.column was used to

separate the components prior to entry into the MS system.

The GC/MS conditions are set out below:

Figure numbers XIII, XIV.

Carrier gas - hydrogen at 1.5 mL/min Temperature range 50-220 deg.0

Vacuum 10-6

Torr. 4 deg./min to scan 350 then 8 deg/min

The mass spectrometer system was adjusted to a

relatively high sensitivity range and calibrated to scan

from 0-350 mass units. The total ion current traces are

shown in Figures XIII, XIV. The components are listed in

Tables V and VI, the corresponding total ion current traces

are shown in Figures XIII and XIV.

253247

311

4

3PS

288 ege GEO 689 740

Csi7 19311 12321 16312C 29:21 22343 tea a:ItS

GS2

SG G 9

•-•

- 49 -

Figure XIII. Total Ion Current Trace Tenax Sample Tube Conditioned at 250 deg. C.

---r- r- • a I 2aa 229 489 Gee CO9 700

0:0 212S 01i7 12:20 12:22 IC: :18 22:44

Figure XIV. Total Ion Current Trace Tenax Sample Tube Conditioned at 300 deg. C.

- 50-

Scan No. Component

43 carbon dioxide

44 carbon dioxide plus unknown HC M. Wt 96

86 toluene

102 chloroethylene

176 monoterpene

204 monoterpene

206 monoterpene.

215 trimethyl benzene isomer

229 monoterpene

242 monoterpene M. Wt 134

249 monoterpene M. Wt 136

253 monoterpene

272 monoterpene

301 unknown

314 hydrocarbon

327 undecane - C11 hydrocarbon

364 menthofuran M. Wt 150

391 myrtenal

406 unknown - possibly a terpene

415 dodecane M. Wt 170

Table V. Compounds Eluted from Tenax Sample Tube Conditioned at 250 deg.C.

Scan No. Component

44 carbon dioxide

249-270 unresolved mixture of trace quantities of unknown mixture

451 unsaturated C10 aldehyde

516 decenal

539-585 unknown, mixture

598 trimethyl naphthalene

619 unknown mixture containing one branched HC

647 unknown ketone

649 heptadecene M. Wt 238

652 heptadecane M. Wt 240

655 branched HC - weak parent ion

679 octadecene M. Wt 252

682 octadecane M. Wt 254

712 nonadecane? or ecosane?

732 palmitic acid (carboxylid acid peaks 60 & 73)

Table VI. Compounds Eluted from Tenax Sample Tube Conditioned at 300 deg.C.

- 51 -

Discussion.

The results indicate that quantitative desorption does

not occur when the Tenax sample tube is heated to 250 deg.0

for trapped terpene components. The traces obtained

indicate that either a higher conditioning temperature, or,

a longer conditioning time is required to reduce the blank

contamination carry over level. Higher desorption

temperatures should improve the initial recovery percentage,

simultaneously reducing the conditioning required provided

that unacceptable levels of thermal breakdown products are

not generated. Desorption temperatures of 270 deg.0 and 290

deg.0 were used by Pellizzari [56] and Bertsch [19] for

recovery of components adsorbed onto Tenax.

A paper by a research group of the U.S. Air Force [192]

lists benzene as a major thermal breakdown product of Tenax

and as a result use a conditioning and desorption

temperature of 240 deg.C. The trace at 300 deg.0 (Figure

XIV) did not show any measurable quantity of benzene, this

would appear to contradict the work of the above authors who

claimed that the quantity of benzene trapped was directly

related to the conditioning temperature in excess of 250

deg.C. Tenax used in allthe traps packed had previously

been conditioned in this laboratory at 325 deg.0 under a

stream of high purity nitrogen for twelve hours. Treatment

at this temperature removed any loosely bound or unreacted

solvents and decreased the tendency to form voids in the

sample tubes. Glass Tenax analytical GC columns showed a

- 52-

tendency to form large visible voids unless the packing was

preconditioned in the above manner. Presumably the above

authors did not precondition at a temperature higher than

240 deg.0 and this may account for the continual loss of

benzene. At a desorption temperature of 240 deg.0

quantitation of components would also be somewhat difficult,

unless, a range of internal standards was added to the

original sample to enable collection efficiencies to be

calculated.

Conclusions.

Air sampling of volatile organic compounds by the use of

a Tenax sample tube should be undertaken with due regard to

the possible effects of conditioning temperature and time.

Care should be exercised in the initial preconditioning of

the packing material. A blank run should be conducted on

the tube prior to dispatch to the sampling area and a

similar blank tube should accompany the sample tubes.

Sample tubes should be stored individually in glass

stoppered carrying containers with a small quantity of

dried silica gel. Where possible Tenax sample tubes should

be allocated to one particular sample type; i.e. one set of

tubes for ambient air monitoring, one set for volatile

terpenes etc.

The use of glass lined metal tubing for Tenax sample

tubes provides the mechanical strength of steel with the

inert properties of glass and has proved most satisfactory

% Inte

nsit

. tim'aMnV3.

Explanatory Diagram of MS Total Ion Current Trace.

- 53 -

for field sampling. The procedure adopted to reduce cross

contamination of sample collection tubes involved the

following: the GLT was baked in a furnace at 600 deg.C,

packed with the preconditioned Tenax, and reconditioned at

300 deg.0 immediately before despatch to the sampling point

in glass stoppered tubes. The sample trap could be stored

for up to two weeks in a cool room without loss of sample,

however processing was generally undertaken as soon as

possible following collection of the sample.

Note. Interpretation of MS total ion current trace. The information from the ion current scans is stored in the data system and the output intensity is calculated relative to 100 percent of the highest peak - unless overloading occurs. The output trace is percentage intensity (vertical axis) vs scan number with time in decimal minutes printed below (horizontal axis). Prominant peaks are labelled with the respective scan numbers (X1,X2,etc). Details of the MS equipment used are provided in Table VII, and in the appendix.

- 54 -

CHAPTER IV

AN EXPERIMENTAL COMPARISON OF VOLATILE ORGANIC

SAMPLING TECHNIQUES.

Introduction.

Exposure to mixtures of volatile organic components has

become an accepted part of normal living experiences and

little attention is paid to various odours unless they are

particularly obnoxious or dangerous. Irritant effects at

low concentration in the air of working environments can be

tolerated with some compounds but not with others.

Assistance with the qualitative determination of an

irritant that was present in-the atmosphere surrounding a

furnace was requested from industry. The persistant, mildly

lachrimatory, eye irritant nature of the substance made

working conditions in the vicinity of the furnace

particularly unpleasant. Conventional methods of detecting

the compound type, i.e. by use of commercially available gas

detection equipment (Draager Tubes etc.) had failed.

This request provided an ideal case study to compare

four different sampling and concentration techniques:-

a) Direct gas sample collection in a Teflon bag

b) Solvent adsorption

, c) Cryogenic trapping

d) Tenax sample tube adsorption.

- 55 -

A. Experimental

The furnace consisted of a rotating steel drum heated to

180 deg.0 by a liquified petroleum gas flame. Modifications

had been made to the burner upon the recent installation of

the unit. Heat loss from the gas flame was minimised by

three different types of thermal insulation that were placed

on the inside of the support structure adjacent to the flame •

area. Hot air from the flame jet rose around the outside of

the rotating furnace and was conveyed by a duct to the

outside of the building. Round the front of the rotating

drum of the furnace was a rubber flap to minimise heat loss,

however, a certain amount of hot gas escaped at this point

and thus carried quantities of the irritating component into

the atmosphere of the room. (Figure XVa) Considerable

discomfort caused by irritation of the face skin, and

lachimatory action to the eyes, occured whilst sampling the

hot gas stream. The effect was somewhat similar to that of

low concentrations of acetyl chloride.

Figure XVa. Front Elevation of Furnace.

1.Gas Flame 2.Glass Fiber Insulation 3.Rubber Seal 4.Rotating Cylinder

- 56-

i) Sampling

All glassware used was cleaned by washing with

detergent, rinsed with distilled water and then heated to

600 deg.0 in a furnace. The solvent used was redistilled

AR. dichloromethane, and where direct adsorption was not

possible Teflon tubing was used to convey the gases.

a) Direct Gas Sampling

Gases escaping through the top of the rubber seal round

the drum of the furnace were sampled directly into a 300 x

300 mm Teflon gas ample bag and the bag sealed with a Teflon

faced septum. This was accomplished by placing the empty

Teflon bag into a 20L plastic barrel with the inlet of the

bag projecting through the lid of the barrel. Air was

withdrawn from the barrel. The resulting decrease in

pressure expanded the bag inside the barrel and allowed the

collection of several litres of sample.

b) Solvent Adsorption

A Venturi water pump was used to draw the sample gases

through two Dreschel bottles in series containing a total of

200mL of the dichloromethane solvent. The Dreschel bottles

were immersed in a water-ice mixture to prevent rapid loss

of the solvent due to evaporation, and gases were ducted to

the system via Teflon tubing. The flow rate was 30L per

hour and sampling continued for two hours.

- 57 -

c) Cryogenic Trapping

A sample of the gas mixture was drawn through a glass U

tube immersed in liquid nitrogen within a Dewar flask to

condense volatile material. The U tube was washed out with

dichloromethane at frequent intervals to ensure that as much

as possible of the condensed organic material was retained.

The volume of gas mixture sampled was 20L. Problems

encountered included frequent ice buildup in the U tube

resulting in blockage of the flow line, and the difficulty

of disconnecting and reconnecting tubing that was rigid at

the low temperature.

d) Tenax Sample Tube

Gases emanating from the furnace were at a temperature

in excess of one hundred degrees, therefore, in order to

retain volatile components on the Tenax sample tube, a

method of cooling was required. This was accomplished by

inserting the sample collecting tube into a water jacketed

length of 8mm diamter copper tube surrounded by a cooling

water jacket (Figure XVb). The temperature of the glass

Tenax tube was therefore maintained at less than twenty

degrees enabling the adsorption of low boiling point

components. The sample tube was held in position by a 6mm

Swagelok fitting silver soldered onto the end of the copper

tube.

-5 8-

dormammumar umwm. ammairmamnrAmmaci

•

• • tf

• / /

5. _

Figure XVb. Water Cooled Sample Collection Probe (Cross-section).

1. Tenax sample tube. 2. Hot gaseous sample in. 3. Cooled gas out. 4. Cooling water inlet. 5. Cooling water •outlet. __6. Cooling water jacket. 7. 6mm Swagelok coupling. 8. Aluminium foil wrapped over

fiber glass insulation.

The inlet of the Tenax sample tube, in the water cooled

jacket, was suspended above the furnace adjacent to the

rubber seal. A flow rate of 50mL per minute was maintained

through the sample tube for two hours.

ii) Analytical results

Analytical work on the collected samples was undertaken

using packed columns on the GC and capillary columns on the

GC/MS runs. Sample pretreatment of the solvent collected

samples consisted of careful evaporation under a controlled

stream of high purity nitrogen gas at 30 deg.C.

a) Direct Gas Sampling

A 2.0mL portion of the gas sample was injected onto a

packed GC column (3 percent Dexil 300 on 100-120 mesh

Chromosorb W HP). A gas tight syringe was used to transfer

the sample to the 2m column maintained isothermally at 50

deg.C. On maximum sensitivity only one minor multiple peak

of retention time 0.50 seconds was noted. This was

identified by GC/MS as air with a trace amount of propane.

4 .

- 59 -

Solvent Adsorption Sample

The 200mL volume of dichloromethane solvent was reduced

to 100 microlitres by careful evaporation under a stream of

dry nitrogen. Water traces were removed by passing the

liquid through a small column of anhydrous sodium sulphate,

and samples were stored in small volume glass vials with

Teflon seals. An equivalent volume of the distilled

dichloromethane was treated in a similar manner to provide a

blank. Injection of 1.0 microlitre portions of sample onto

the Dexil 300 packed column, described above, gave a GC

trace with a large solvent peak with a number of shoulder

peaks. Trace amounts of other compounds were observed,

however the blank solvent run provided a very similar trace,

indicating the presence of solvent impurities. A high

resolution 55m OV101 SCOT colUmn was installed in the GC

mass spectrometer system to enable separation and, where

possible, identification of the components. 0.5 microlitre

of sample was injected onto the column in a Grob type

injection at ambient temperature. Temperature programming

was commenced at 50 deg.C. Details of operating conditions

are set out in Table VII.

- 60 -

Gas chromatograph: Pye series 204 Column: OV101 SCOT 55m glass 0.5mm dia Carried gas: He at 1.0 mL/min. Temperatures(deg.C): Inlet 225, outlet 250, interface 250 Oven programme: Ambient, then 50 deg. to 200 deg,

2 deg/min to scan 375 then 8 deg/min. Mass Spectrometer: VG Micromass 7070F

Scan rate 1 per 2.5 seconds. Energy 70Ev.' Data System: VG 2235 using a PDP 8/A620 minicomputer.

Table VII. GC/MS Operating Conditions

Identification was made by comparison of mass spectral

data in the literature [193], and the library of authentic

samples stored in the data system, and also on the basis of

retention time data. Additional information was gained from

the fact that OV101 is a relatively non-polar phase and

separates most components on a boiling point basis. The

trace obtained from the solvent run is shown in Figure XVI

and the component identities in Table VIII.

1 483157

tetrachloroethyle

ne 9.

chloroform 3.

• a IVO ZOO 228 480 423.21 01 0 111 8.1 1210 . 1C.3 ZOI1 Z3.0 7V0 r7:

tric

hlo

roethylene 6.

1 8

dichlorome

thane 1.

tric

hloroethane 4.

C.N1

00 L. C 00

"OW

37

-61 -

Figure XVI. TIC Trace Dichloromethane Blank OV101 Capillary Col. 50-200 deg. C. (Conditions as per Table VII.)

Peak No. Scan No. Identity

1 75-90 dichloromethane 2 97 dichloroethylene 3 121 chloroform 4 143 1,1,1 trichloroethane 5 162 cyclohexane 6 188 trichloroethylene 7 234 4 methyl 2 pentanone 8 276 toluene 9 352 tetrachloroethylene

10 432 ethyl benzene 11 444 xylene

Table VIII. Components Identified in Dichloromethane Solvent Blank

0

0 _c

1,1,

1 t

ric

hlo

roe

t han

e 4.

1SS

chlo

rofo

rm 3

.

13

' 1363315

La

hep

tan

al is

om

er 1

7.

1 2.40 288 3:i2 11:13

r 228 Z 488 440 488 17:S8 14:88 isto - 2711-5 13:13

- 62 -

o af,,,--,--77"-77-NT i1 1---T----1 — I-- 0 1-0 m Ira iss zem

0:2 1:33 2315 4:52 clas 87

Figure XVII. TIC Trace of Solvent Trap Volatiles OV101 Capillary Col.. Conditions as per Table VII.

Peak No. Scan No. Identity

1 74-88 dichloromethane*

2 93 dichloroethylene*

3 117 chloroform*

4 139 1,1,1 trichloroethane*

5 150 benzene

6 159 cyclohexane*

7 177 1,2 dichloropropane

8 185 trichloroethylene*

9 228 4 methyl 2 pentanone*

10 258 toluene*

11 284 4 methyl pentanal (?)

12 296 tetrachloroethylene*

13 332 ethyl benzene*

14 337 )

15 350 ) 0,m and p xylene*

16 362 )

17 378 heptanal isomer

18 404 octanal

19 411 n-decane

Table IX. Components Identified in Solvent Trap Concentrate.

Injection of the same quantity of sample from the

-63 -

solvent trap under similar conditions gave the total ion

current trace shown in Figure XVII. Initial oven

temperature conditions caused a shortening of retention

times and hence the peak scan numbers are not identical to

those in Figure XVI. Components identified are shown in

Table IX with peaks also found in the solvent marked with an

asterisk *• High concentrations of dichloromethane and

tetrachloroethylene caused overloading of the MS system and

temporary shutdown of the beam as shown by the return of the

trace to baseline.

c) Cryogenic Trap Sample

The dichloromethane washing from the cryogenic trap

sample also contained a considerable amount of water, was

passed through an anhydrous sodium sulphate column and then

evaporated to 200 microlitres, as described above. The GC

and total ion current traces were virtually identical to the

dichloromethane solvent blank traces. No other compounds

were detected.

d) Tenax Trap

The Tenax sample trap was removed from the stoppered

glass carrying tube and placed into the thermal desorption

apparatus, flushed with carrier gas and the system coupled

to the GC/MS. A 10 minute thermal desorption time at 300

deg.0 was used. The total ion current trace is shown in

Figure XVIII the components are identified in Table X, and

the mass spectra are listed in the appendix. •

14

I I

03

! 13

go

I

I I

• -

--n

acet

oncm

____

tin

ethy

lene

on

ch

lori

de 5

. ca

rbon

dio

xide

7.

n entane meth 1 entane 8

chlo

ro-

form

9.

I benzene 10.-

4-4

•

chloroform 11.

,y

00

acrole

in

aceto

ne

tolu

ene 12.

w te

trachloroethylene 13.

•0

)

CD

••

mono

terpene 14.

phenol

15

n-pe

ntan

e

o.m.

p. c

reso

l 16

. 17

. 18

.

ethy

l ph

enol

19.

dime

thyl

phe

nol

20.

2 et

hyl

phen

ol 2

1. •

n pentadecane 22.

mlhe

ptad

ecan

e 21

.

-65-

Peak No. Scan No.

1 20 2 23 3 24 4 28 5 29 6 32 7 50 8 70 9 75

10 114 11 119 12 245 13 302 14 402 15 438 16 460 17 469 18 476 19 490 20 495 21 502 22 610 23 642

Identity

Hydrocarbon (pentadiene) acrolein + pentadiene isomer + acetone acetone methylene chloride + acetone + unknown methylene chloride Freon carbon dioxide n-pentane + methyl pentane chloroform benzene chloroform toluene tetrachloroethylene unknown monoterpene phenol

) ) o,m and p cresol

) ethyl phenol dimethyl phenol 2 ethyl phenol pentadecane unknown

Table X. Components Identified in Tenax Trap.

High concentrations of contamination adsorbed from the

solvent traps tended to mask some of the earlier peaks,

however, acrolein, acetone , a number of phenols and cresols

were detected. A plot of the 27 and 56 mass ions in the

first 100 scans shows the presence of a suspected acrolein

peak in the early section of the trace - Figure XIXa. The

characteristic ions 27 and 56 maximise at scan number 23

under the leading edge of the acetone peak, this information

coupled with the coincident retention time of acrolein

confirmed the presence of the substance.

- 66 -

B.Discussion.

The problems associated with the various trapping

methods, as outlined in the literature sources, were shown

to be considerable in this attempt at collecting a trace

component in an industrial process. Solvent contamination

and escape proved to influence each determination. Trapping

efficiency of the unknown component in the direct headspace

collection, the solvent trap, and the cryogenic trap, proved

to be too low, or the losses during concentration too high,

that instrumental methods failed to detect the substance.

This was in spite of the fact that the substance was

painfully obvious to the eyes and nose.

Components detected in the solvent trap extract could

not account for the observed effects in the concentrations

measured. The components present could be explained as

either solvent impurities, products from the furnace or

breakdown products of the insulation.

Acrolein (propenal), detected in the Tenax trap at a

concentration -of approximately 1 part per million, has a

toxic dose level (TDL) OF 0.1 PPM [194], and the boiling

point of 52.7 deg.0 correlates with the elution from the GC

column just prior to acetone (bp 56.2 deg.C). A small

quantity of acrolein was manufactured synthetically and

collected along with some of the industrially generated

material. The two substances co-eluted from the GC column

and gave identical mass spectra. (Figure XIXb.) The

physiological effects of the synthetically produced acrolein

- 67 -

were identical to the gaseous product from the furnace; to

this point some difficulty was experienced in convincing the

industrial operators that the active constituent was present

in relatively low concentrations.

A search was conducted of the stored spectra from the

solvent and cryogenic trap runs but acrolein and several of

the other low boiling components could not be detected.

Apparently the evaporation of the solvent and the drying

step has removed the lower boiling point components.

Literature sources describe a number of ways in which

acrolein may be produced, from oxidation of petroleum gas

[195], as a by-product from pyrolysis of paints and

degreasing solutions and from the heating of plastic tubing,

insulation and metal foil tape [196]. Two main sources of

the acrolein would therefore appear possible.

i. from oxidation of the L.P. fuel gas ii. from pyrolysis of the furnace construction/insulation

material.

- 68 -

CD

SS

SO

Figure XIXb. Mass Spectrum of Acrolein (propenal).

- 69 -

C.Summary and Conclusions.

Four trapping techniques were used in order to determine

the nature of an air contaminant in the vicinity of an

industrial furnace. Direct headspace injection provided

little useful information as the ambient concentration of

the contaminant was below the instrument detection level.

Solvent trapping and cryogenic concentration did not provide

sufficient quantity of the unknown contaminant, free from

solvent interferences, to give positive identification.

Major losses of the highly volatile unknown compound

occurred during the removal of solvent and/or water, and

this problem coupled with the low concentration present,

made identification difficult with the more classical

techniques.

Tenax trapping of the component from the air allowed

qualitative and approximate quantitative determination of

acrolein in the hot gas stream from the furnace. Acrolein

was confirmed using a synthetically produced compound as a

comparison. Follow-up work using the more polar Chromosorb

104 as a trapping medium also confirmed the presence of

acrolein at the lppm level.

The source of the acrolein was either:

i) incorrect combustion conditions of the L.P. flame.

ii) a pyrolysis by-product of the furnace construction/ insulation material.

- 70-

CHAPTER V.

VOLATILE COMPONENTS OF SOME NATIVE PLANTS.

Introduction.

A need exists to examine systematically the chemical

composition of the essential oils of Australian native

plants. Work on the Eucalyptus genus has been conducted by

Penfold and Morrison [197], Hellyer and McKern [198] and

others at the Museum of Applied Arts and Sciences in New

South Wales. McKern [202] has reviewed some of the history

of essential oil studies in Australia, and has also reviewed

[203] the problems associated with attempts to utilize

chemical compositions of volatile oils in taxonomy. General

reviews of work on alkaloids and terpenes are published by

the Royal Society of Chemistry [204,205]. Investigations of

the alkaloids of native plants has been conducted by Bick et

al [206], and of terpene composition by Ayling [207].

A number of Tasmanian plants have volatile components

that impart attractive odour or taste properties. Two of

these, Drimys lanceolata and Prostanthera lasianthos, are

examined in this work, with the object of documenting the

steam distillate oil and of developing techniques for the

characterisation of small quantities of volatile organic

compounds. The third section examines the relationship

between the volatile components of Boronia meciastigma, a

native plant of Western Australia.

-71 -

A. Components of the Steam Volatile Oil of

Drimys lanceolata.

Introduction.

Drimys lanceolata occurs in the Tasmanian highlands and

also in Victoria and New South Wales. Commonly called

'Mountain Pepper' due to the very 'hot' peppery taste of the •

leaves, the plant is widely distributed in Tasmania. Plants

on high windswept plateaus and exposted ridges tend to be

short and flattened in appearance, whereas, in gullies the

growth tends to resemble small trees or tall shrubs. The

height varies from 0.2m to 5.0m and the young stems are

crimson in colour [208].

Previous experimental work on extracts from the plant

were conducted by Stevens [209], and Loder [210], and work

on the related New Zealand plant, Pseudowintera colorata,

was performed by Corbett and Grant [211].

A preliminary investigation of the steam distillate of

Tasmanian samples of Drimys lanceolata by Stevens indicated

that there existed considerable variations in physical and

chemical properties of oils derived from plants found in

separated areas of the state. The sesquiterpene alcohol

guaiol was found to crystallize from two of the oils.

Loder [206] used a hexane extraction method to isolate

two crystalline components from the leaves, Rolygodial

(0.26%) and guaiol (0.035%). Polygodial was described as

- 72 -

partly responsible for the pungent taste of the leaf.

Corbett and Grant listed a number of components derived from

column distillation of the volatile oil from Pseudowintera

colorata (Maori Pepper). The fractions and percentage

composition are listed in the discussion section.

The composition of the volatile leaf oil components of

Drimys lanceolata has apparently not been reported in the

literature. Samples of the plant were collected from a

number of areas of Tasmania to determine whether significant

compositional variations occured.

1) Experimental

a) Sample Collection

Small branches and leaves were collected from five

scattered areas of Tasmania. Samples of the plant collected

adjacent to the Mt. Wellington park differed from the others

in that the height of plant was relatively small (0.25m).

The other samples were collected from small trees or shrubs

growing in sheltered valley situations in deeper soil.

b) Distillation

The leaves were stripped from the branches and the steam

volatile components were passed through a vertical glass

column packed with 5mm lengths of glass tube and condensed

before collection in a saturated aqueous sodium chloride

solution. The oil was separated from the aqueous solution by

decantation and triple extraction with diethyl ether. Ether

- 73-

was removed by low temperature evaporation using a dry

nitrogen gas stream. Residual water was removed by passing

the oil through a small column of granular anhydrous sodium

sulphate. Percentage yield and some physical properties of

the volatile oil are recorded in Table XI.

Sample Plant Oil Area Height(m) Yield (%) Density(g/mL)

Mt. Wellington 0.25 0.37 0.901

Parrawe 2.5 0.70 0.910

Mt. Field 2.5 0.43 0.917

Moogara 1.5 0.50 0.920 Wtern Tiers 2.0 0.35 0.928

Table XI. Average Percentage Yield and Physical Properties of the Steam Distillate of Drimvs lanceolata.

c) Analytical Results

Identification of components in the steam distillate was

carried out by comparison of GC retention times on two

columns and mass spectral data. The result for each

district is derived from a composite of the oil from three

trees.

The GC traces are shown in Figures XX - XXIV. Relative

retention indices were caculated from separate and -

coinjected mixtures of C10 to C22 n-alkane hydrocarbon

standards at a concentration of 0.040 percent each in

chloroform. Reproducability of the coinjected mixture was

generally excellent on the OV101 capillary GC column;

however, some variations occured on the Carbowax 20M column

compared with the literature values [212]. Retention times

and peak areas were recorded on a Shimadzu Chromatopac-E1A

- 74-

and the percentage composition of each component calculated

on the basis of the peak areas.

The GC/MS total ion current traces (Figures XXVI,XXVII)

were obtained on the VG instrument previously described,

using a 50m CW2OM silica capillary GC column (Figure XXVII),

and a similar OV101 column (Figure XXVI); temperature

programmed at 4 deg.C/min. from 80 to 200 deg. C. Individual

component mass spectra are listed in the appendix at the end

of the thesis.

The peak scan numbers and component identities of the

more complex Moogara sample are listed in Table XII.

_ I A---

1 0

i_

.

0 0 -

._

19

•

.

24 .

CD

.

._ (D 7

35..

1 r

.

.

i

15

- 75-

Time (mins)

Figure XX. GC Trace D. lanceolata on CW2OM Capillary Col. 50-200 deg. C at 2 deg./min. Sample ex Moogara. Component Identification Table XII.

Time (mins) -4-

Figure XXI. GC Trace D. lanceolata on CW2OM Capillary Col. 50-200 deg. C at 4 deg./min. Sample ex Parrawe. Component Identification Table XIII.

Fl D

Resp

ons

e ÷

15

3 9

21 2-

36

3 0

22

15 2 4

2 5

26

36 30

1 k_

Time (mins)

F I D

Resp

o nse

+

- 76 -

Time (mins)

Figure XXII. GC Trace D. lanceolata on OV101 Capillary Col. 50-200 deg. C at 4 deg./min. Sample ex Wtern Tiers.

Component Identification Table XIII.

Figure XXIII. GC Trace D. lanceolata on CW2OM Capillary Col. 50-200 deg. C at 4 deg./min. Sample ex Mt Field. Component Identification Table XIII.

F I D

Rep

onse

±

- 77 -

15 24

36

313

,

Time (mins) -4-

Figure XXIV. GC Trace D. lanceolata OV101 Capillary Col. 50-200 deg. C at 4 deg./min. Sample ex Mt Wellington.

Component Identification Table XIII.

CO7

•-■

63- 1 i.

100 200

----r16llie - XXV.- 6-614&1' Mass Spectrum of - Guatol ex Wtern Tiers.

FID

Res

ponse

-+

S8 1 476

S00 2510

1000 31125

— ...... GOO 113:S1

r- .030

G:17 12:34

31.33 00 000

1S.40

- 78 -

0.1

Figure XXVI. TIC Trace D. lanceolata ex Wtern Tiers. OV101 Capillary Col. 80-200 deg. C; 4 deg./min. Component Identification Table . XIII.

Figure XXVII. TIC Trace D. lanceolata ex Moogara. CW2OM Capillary Col. 80-200 deg. C; 4 deg./min. Component Identification Table XII.

cs*., cr.)

163360

99

352

140

- 79-

Peak Scan Retention Percent Identity No. No. Index Composition

solvent (chloroform) ,c-pinene /s-pinene myrcene -ic-phellandrene limonene /3- phellandrene 1,8 cineole monoterpene (?) p cymene unknown X (base 68) isomer of X isomer of X cubene

unknown bourbonene

unknown unknown (X + CH2) linalool caryophyllene terpinene-4-ol sesquiterpene monoterpene alcohol

-,c-terpineol sesquiterpene piperitone cadinene ( ?) cadina 1,4 diene calamanine farnesol unknown methyl eugenol (type) sesquiterpene alcohol sesquiterpene alcohol guaiol oxygenated sesquiterp. eugenol isomeric guaiol (?) unknown methyl eugenol + CH2 unknown unknown myristicine (?) unknown others

Table XII. Identification Details of Steam Volatile Oil of Drimys lanceolata ex Moogara.

1 96 - - 2 98 1039 7.8 3 116 1124 3.6 4 127 1156 1.5 5 130 1177 0.3 6 141 1206 1.9 7 145 1218 0.1 8 148 1228 18.8 9 158 1246 0.1

10 170 1272 0.4 11 206 1328 0.3 12 224 1351 0.4 13 268 1402 0.1 14 279 1420 0.3 15 304 1448 0.1 16 325 1482 . 0.1 17 332 1494 0.1 18 342 1501 0.3 19 353 1506 38.8 20 386 1617 0.4 21 395 1628 0.4 22 435 1648 0.4 23 455 1655 0.1 24 476 1661 3.4 25 481 1702 0.2 26 503 1739 0.3 27 516 1758 0.8 28 537 1782 0.1 29 581 1842 3.6 30 605 1875 0.1 31 666 1938 0.2 32 738 2044 0.1 33 771 2046 0.2 34 779 2049 0.2 35 801 2058 8.8 36 826 2078 0.4 37 858 2103 0.4 38 861 2112 0.3 39 874 2122 0.1 40 885 2132 0.3 41 905 2154 0.1 42 910 2158 0.3 43 929 2172 0.5 44 1015 2208 0.1

3.2

- 80 -

Peak Component No

Mt. Parrawe Wellington

Mt. Field

Western Tiers

2 .0c-pinene 10.2 5.4 5.1 6.5 3 /3-pinene 11.2 1.3 4.6 8.1 4 Qc-phellandrene 3.1 3.9 5.8 0.7

• 5 myrcene 0.6 0.2 1.6 3.7 6 /3-phellandrene 0.4 0.1 0.1 ) 7 heptanone 0.1 0.1 0.1 ) 1.0 8 limonene 0.1 0.7 5.7 0.6 9 1,8 cineole 2.6 0.5 11.2 7.6

10 monoterpene 0.1 0.1 1.1 0.1 11 p-cymene 0.2 1.1 1.9 0.3 12 /3-ocimene or,

A-3 carene 0.1 0.1 0.1 0.8 13 terpinolene 0.6 0.3 0.7 0.2 14 unknown 0.4 0.1 0.1 0.3 15 linalool 31.8 40.7 18.8 17.2 16 monoterp. acetate 1.2 0.3 0.1 0.1 17 caryophyllene 0.1 ) 0.1 0.1 18 terpinen-4-ol 0.7 ) 2.9 0.4 0.9 19 .-terpineol 4.1 0.9 0.8 2.3 20 sesquiterpene 0.3 0.1 - - 21 piperitone 0.1 0.9 7.0 3.4 22 safrole 0.1 0.2 1.1 1.7 23 unknown 2.7 0.1 0.8 0.3 24 eugenol 8.6 5.7 10.0 5.5 25 methyl eugenol 1.6 0.9 1.3 0.7 26 unknown 0.3 0.2 1.1 0.1 27 unknown 0.2 0.6 0.1 0.3 28 geranial 0.5 0.4 0.4 0.1 29 unknown 0.2 2.4 1.1 0.5 30 myristicine 1.2 5.7 5.0 5.1 31 /g-cedrene(?) 0.1 0.1 0.1 0.1 32 x-murolene(?) 0.1 1.4 1.7 0.3 33 mycenol or )

elemol ) 0.2 0.9 2.2 1.1 34 /3-farnesene(?) 0.1 3.1 0.9 0.3 35 dihydrocarvyl

acetate(?) 0.9 1.8 0.6 1.3 36 guaiol 4.3 8.0 1.6 16.5

Others 10.8 13.0 6.7 12.2

Table XIII. Percentage Composition Drimys lanceolata From Various Areas of Tasmania.

The percentage composition of the oil samples from the

other areas of the state are shown in Table XIII.

A number of components were not identified due to the

-81 -

lack of comparative data in the literature, and the lack of

time available to prepare pure standard samples from the

crude oil on a preparative scale.Characterisation of minor

components would require the collection of large quantities

of leaf material as the percentage of oil present is

relatively low.

ii) Discussion

Variation in the percentage composition of the main

components: linalool, -e-pinene,/1 -pinene, 1,8 cineole,

guaiol, was considerable (up to three times for each

component) between areas. The sample taken from Moogara

varied in composition from the other sites in that the oil

contained a number of components that were not present in

significant concentrations in samples collected in the other

areas. The presence of a component with a similar mass

spectrum to guaiol was of particular interest (mass spectra

from Scan No. 861 compared with Figure XXV). Unfortunately

there was insufficient material to isolate and confirm the

structure of this component.

The similarity in composition of the Tasmanian plant to

that of New Zealand Pseudowintera colorata is shown by the

components from this plant listed in Table XIV.

-82 -

Fraction % Composition Components 1 1.0 ,..e-pinene, -1n-thujene 2 1.0 /3-pinene 3 0.5 myrcene 4 0.5 carbonyl cpd 5 2.25 limonene, dipentene,Qe -terpinene 6 2.25 /g-phellandrene mixture 7 1.0 terpene alcohol (C io HnO) 8 - carbonyl cpd (0 10 H u+ 0) 9 1.0 --e-terpinene

10 1.0 terpinolene,,4 7-cymene 11 0.5 ester 12 0.5 alcohol (CO 34 0) 13 1.0 alcohol (C,

0 H.,0 0) .

14 0.5 hexenyl yalerate 15 1.0 eugenol 16 16.5 bicyclic sesquiterpene 17 1.0 bicyclic sesquiterpene 18 1.0 aromadendren 19 1.0 humulene 20,21 4.5 alcohol unknowns 22 3.0 aromatic hydrocarbon 23 4.5 alcohol 24 2.0 sesquiterpene ketone (C,5 Hz.t 0)

Residue 52.0 tricosane, pentacosane and sesquiterpene ketone

Table XIV. Percentage Composition of Steam Distillate of Pseudowintera colorata [211].

A considerable amount of additional experimental work is

required to enable a final characterisation and confirmation

of the identity of the remaining components of the steam

distillate of the oil of Drimys lanceolata. Techniques such

as GC Fourier transform infrared analysis, as well as

laser-Raman and UV methods, for determining functional

groups and molecular structure would assist in the complete

characterisation of the oil.

- 83 -

B. Components of the Steam Volatile Oil of

Prostanthera lasianthos.

Introduction.

Prostanthera lasianthos occurs in Tasmania, Victoria and

New South Wales on the margins of wet eucalypt forests. The

shrub or small tree grows to a height of from 2 to 6m [208].

The strong-smelling flowers are formed in December to

January, hence the common names of Christmas Bush (or Mint

Tree).

The terpene composition of the steam volatile oil of the

related Prostanthera rotundifolia has been reported by

Ayling [207] but there was no indication in the literature

of previous experimental work being performed on P.

lasianthos.

1) Experimental

(a) Sample Collection

Leaves were collected in April from four trees growing

in small valleys draining to Pirates Bay on Tasman

Peninsula. The leaves were concentrated on the extreme

outer ends of the branches and were somewhat low in numbers

due to heavy growth of surrounding plants. Height of the

trees were 3 to 4m.

- 84 -

(b) Distillation

The procedure followed for distillation of the leaf oil

was as described in Section A for D. lanceolata. Percentage

yield (by weight) of the steam distilled oil derived from a

composite of the leaves from the four trees was 0.12.

(c) Analytical Results

50m silica capillary GC columns were used to

separate components in the oil and their mass spectra

recorded. The total ion current trace (Figure XXVII) was

obtained by injecting 0.4 microliters of the oil (with a

10:1 split), into the GC/MS using a CW2OM GC column

temperature programmed from 50 to 210 deg.0 at 4 deg./min.

to separate and characterise the peaks. Component

identification was based on relative retention index and

mass spectral data. The component mass spectra from two

GC/MS runs are shown in detail in the appendix. DE1036

(Figure XXVIII) shows the total ion current trace from the

OV101 silica capillary GC column with a similar loading to

the CW2OM column but temperature programmed from 50 to 240

deg.0 at 4 deg./min. Scan numbers and tentative MS

identification data are recorded in Table XV, and the

component mass spectra are listed in the appendix.-

The n-hydrocarbon standards peaks are shown in the

temperature programmed GC trace of Figure XXIX. Table XVI

lists the standards and the temperature programmed retention

times used to calculate the retention indices obtained on a

- 85 -

50m OV101 glass SCOT capillary column. The same column

under identical programming conditions was used to obtain

the GC trace of Figure XXX. The retention indices of the

recorded peaks, the literature values and the percentage

composition are tabulated in Table XVII.

377

71060

254

7B

296

47

- 86 -

CRL: 11)550 16628

72

85

7?1

631

4 581

147 9 2

9:1 G137 E:80 19.58

008 2.8126

imm 17113 Scan No 23:2 83:28 Time

Figure XVII. TIC Trace P. lasianthos CW2OM Capillary Col. 50-210 deg. C at 4 deg./min. Component Identification Table XV.

280 408 288 21% 2:i0 12:2C 18:53

880 2Ss I) Izo8 Scan No. 11/.4s

Time

Figure XVIII. TIC Trace P. lasianthos OV101 Capillary Col. 50-240 deg. C at 4 deg./min. Component Identification Table XV.

- 87 -

60 70

C9

C10

C11 C17 C3 C12 C16 C14 C15 F

ID R

esp

onse

.

C20 C18 C19

20 40 Erne (mins.) . .

Figure XXIX. GC Trace n-hydrocarbon Standards. Conditions as Shown Below.

Standard Retention Time (minutes)

Retention Index

n-C9 8.55 900 n-C10 13.50 1,000 n-C11 19.53 1,100 n-C12 25.95 1,200 n-C13 32.36 1,300 n-C14 38.56 1,400 n-C15 44.51 1,500 n-C16 50.20 1,600 n-C17 55.61 1,700 n-C18 60.81 1,800' n-C19 65.78 1,900 n-C20 70.58 2,000 n-C21 75.69 2,100

Table XVI. Retention Times and Indices for n-hydrocarbon Standards on OV101 Glass Capillary Column 50-220 deg.0 at 2 deg./min.

- 88 -

14

33 47

32

28

19

8

10 20 30 40 50 60 70 80 85 Time (nuns.) --

Figure XXX. GC Trace P. lasianthos OV101 Capillary Col. 50-220 deg. C at 2 deg./min. Component Identification Table-XV.

- 89 -

Scan No. Scan No. Peak

CW2OM OV101

No. Tentative Identification

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

85 78 123

153-189 243 250

( 254 146-180 ( 267

( 296 ( 309

327 304,319 331 258

344 292 377

394 423 484

404 511

420 581

437 458 460 492

594

499 505

693 514 608 617 631

532 658 677 691 721

548 735

890, 852

584 593-603

741 742 796 801-832 833

655 1123

670 714-765

845 857-976

978 992

1002 1052

1019

Table XV

solvent 4 methyl-pent-1-en-3-one unknown pentyl ester benzaldehyde

-pinene camphene /5 -pinene myrcene < -phellandrene cymene mixture limonene 1,8 cineole + cymene 1,8 cineole unknown momoterpene trans hex-3-enol cis linalool epoxide trans linalool epoxide linalool unknown unknown camphene hydrate pinocarvone unknown archillinol (?) fenchone (?)

caryophyllene terpinene-4-cd sesquiterpene (farnesene?) unknown unknown mixture ..6-terpineol .-selinene 4 phenyl butan-2-one carvone unknowns unknown

elemene myrtenol unknown carveol iso-bornyl acetate thymol unknowns p-cymene-8-ol unknowns unknown ledol /3-eudesmol

Component Scan Numbers and Tentative MS Identification of Prostanthera lasianthos Oil Components.

- 90 -

Peak Rel.Ret. Rel.Ret. Percent

No. Index Index Composition (Lit.) (Calc.)

1

Component Identity

solvent 2 0.1 4 methyl-pent-1-en-3-one 3 1.3 unknowns 4 0.1 pentyl ester 5 947 0.2 benzaldehyde 6 942 930 3.2 .4:-pinene 7 954 941 0.8 camphene 8 . 981 968 1.8 /3- pinene 9 986 987 0.5 myrcene

10 1002 996 0.7 ,?< - phel 1 andrene 11 1020 ) cymeme mixture 12 1030 ) 1024 22.6 limonene 13 ) 1,8 cineole + cymene 14 1027 ) 1,8 cineole 15 0.1 unknown 16 0.1 trans'hex-3-eno1 17 1068 0.1 cis linalool epoxide 18 1082 1080 0.2 trans linalool epoxide 19 1092 1093 2.1 linalool 20 1101 0.1 unknown 21 1107 0.1 unknown 22 1111 0.2 camphene hydrate 23 1125 2.4 pinocarvone 24 1138 1.1 unknown 25 1152 1.6 archillinol 26 1080 0.3 fenchone ? 27 1428? 1351 0.2 caryophyllene ? 28 1175 1165 3.2 terpinene-4-ol 29 1447 0.2 sesquiterpene (farnesene?) 30 1467 0.1 unknown 31 0.3 unknown mixture 32 1185 1180 3.8 *Ia-terpineol 33 1494 3.9 /s-selinene 34 1502 1.8 4 phenyl butan-2-one 35 1228 0.4 carvone 36 1.7 unknowns 37 0.2 unknown 38 0.8 ).-elemene 39 0.4 myrtenol 40 0.4 unknowns 41 0.7 carved] 42 1279 1275 0.2 iso-bornyl acetate 43 1287 1.3 thymol 44 0.3 unknowns 45 1.1 p-cymene-8-ol 46 3.7 unknowns 47 1581 3.4 unknown 48 1623 1.4 ledol 49 1728 1.9 /3 -eudesmol

28.9 other Table XVII. Component Identification by Retention Index

(on OV101) and Approximate Percentage Composition Prostanthera lasianthos.

- 91 -

ii) Discussion

The use of retention indices to assist in the

identification of components in complex mixtures has been

widely reported in the literature [213, 214].

Identification of a well resolved component in a GC run is

relatively straight forward if the component retention time

has been accurately measured previously and the run is

reproduceable. Problems are encountered when the peak is

not well resolved or, if the component has not been

previously measured. Tables provided in the literature

[212] are often of assistance but must be used with caution.

Polar phases, such as Carbowax, can readily alter in

characteristics as the column ages. Components that eluted

in the order a,b,c,d,e can alter and elute a,b,d,e after a

certain amount of the liquid phase has been stripped from

the column with either repeated injections of solvent, or

high temperatures.

The presence of a large number of components with

similar boiling points made baseline separation and

quantitation difficult. Hence, quantitation of components

by area integration proved to be somewhat inexact. Main

components were 1,8 cineole and a mixture of cymenes, obc-

pinene,.4-terpineol, terpinene-4-olOselinene and an unknown

compound with a molecular weight of 220. A relatively high

proportion of the unknowns were oxygenated sesquiterpene

compounds that would contribute to the aroma characteristics

of the oil. Preliminary separation of the compounds into

-92-

terpene hydrocarbons and oxygenated classes would assist in

clarifying the GC trace and allow greater accuracy in

quantitation of components.

Ayling [207] examined the steam volatile oil of the

related Prostanthera rotundifolia. A comparison of the

composition of the main components of this oil with

Prostanthera lasianthos is given in the following table.

Component

.04 -pinene

P. lasianthos P. rotundifolia [207]

3.2 1.4 camphene 0.8 0.1

-pinene 1.8 2.3 myrcene 0.5 ) 'le -phellandrene 0.7 ) 3.7 limonene ) 1.1 1,8 cineole ) 22.6 47.0 p-cymene -. 23.2

terpinen-4-ol 3.2 1.5 cic -terpineol 3.8 4.1 terpene alcohol 1.1 6.9 other 62.3 8.7

Table XVIII. Comparison of the Percentage Composition of Prostanthera lasianthos and P. rotundifolia.

1,8 cineole is the major component in both oils and many

of the minor components are present in somewhat similar

concentrations. Large differences are however apparent in

the higher boiling components of the oils.

iii) Conclusions.

Examination of the steam volatile oils of D. lanceolata

and P. lasianthos provided the opportunity to develop

retention index and mass spectral techniques applicable to

volatile components. The advantages of fine bore silica

capillary columns and the introduction of the 'end of the

- 93 -

column through to the ion source of the mass spectrometer

were proved using the steam volatile oils.

- 94 -

C. Volatile Components of Roronia megastiqma.

Introduction.

The essential oil industry in Tasmania has developed

gradually over the last few decades into a relatively small

but viable agricultural/processing industry. Recent

, research within the Agricultural SciencelFaculty __I of the

University has indicated that a number of groups of plants

produce high quality essential oils when grown in selected

areas of Tasmania [215].

The determination of the optimum time for picking the

leaves or flowers of a crop is an important factor in the

production of a high quality oil. Collection of the

volatile compounds from the surrounding air and rapid

analysis for critical components may provide a viable

alternative to the normal steam distillation procedure. The

collection of headspace air samples has a number of

advantages in that it provides a non-destructive collection

method and under most conditions an inbuilt averaging system

as the sample collection tube can be moved from one area of

a field or glasshouse to another. An automated variation of

this collection-analysis system would provide rapid

analytical results for the harvester.

Commercial processing of amniA_EllegAatizo involves

extraction of the soluble components by solvent and the

production of a 'concrete' consisting of volatiles and

higher molecular weight substances [216]. The objective of

- 95 -

the work described below was to compare the headspace

components trapped from the flowers, the headspace above the

concrete and the components present_in the concrete.

i) Experimental

a) Sample Collection

Boronia flowers (Boronia megastiqma) were provided by

Mr. G. Leggett from the Agricultural Science Faculty.

Samples of the headspace above the flowers were obtained by

placing the flowers (163), in a clean glass jar (previously

baked at 500 deg.C) and drawing the volatiles into a Tenax

sample collection tube at 40 mL per minute for 15 minutes.

The flowers were then extracted with petroleum ether and

the solvent was evaporated by warming the solution and

blowing a stream of dry nitrogen over the surface. The

larger portion of the resulting waxy residue - concrete -

was then heated to its softening temperature (45 deg.C) by

immersing the glass container in a water-bath. Headspace

from the concrete was collected using a second Tenax tube

under similar conditions to the first sample. A portion of

the concrete was directly injected into the GC/MS system for

comparison with the headspace volatiles.

b) Anablitical Results

' Separation of components was achieved using a 50m glass

OV101 SCOT capillary GC column. Components were identified

by mass spectral information verified by comparison with

- 96 -

library spectra of authentic compounds from previous work

[215], and by retention times. Individual mass spectra are

presented in the appendix.

Gas chromatographic conditions are described in

Table XIX, and component identification in Table XX.

Concrete Concrete Headspace

Flower Head space

Designation AG110-AG113 DEBRO4 DEBROO Collection Temp.(deg.C) 45 20 Injection In Solvent Splitless Splitless Initial GC Temp. 80 30 30 Final GC Temp. 250 200 200 Carrier Gas He He He

Flow Rate (mL/min) 1.0 1.5 1.5 Inlet Temp. 200 200 200 Outlet Temp. 250 250 250 Temp.Prog.Rate (deg.C/min) 4 3 3

Table XIX. Gas Chromatography Conditions for Boronia megastigma.

3.

1.2 B

18.

6 152 ZS 21 SI h78 gr3 141Mk

8.8

19S3

1 2

Figure XXXI. TIC Trace Headspace of Boronia megasticima Flowers. OV101 Capillary Col. Peak Identification Table XX. _

- 97 -

(1)

Figure XXXII. TIC Trace Headspace of Boronia meciasticima Concrete. Collected by Tenax Sample Tube. OV101 Capillary Col. Peak Identification Table XX.

/so 8CAN MITER 23.14 Ret. Time (m 0.1

- 98 -

Figure XXXIII. TIC Trace of Concrete of Boronia megasticima [215]. OV101 Capillary Col. Peak Identification Table XX.

- 99 -

Peak Component No.

Code:

Concrete

Ag

Scan Number Concrete

Headspace DEBRO4

Flower Headspace DEBROO

la. Unknown monoterpene 8 340 1. -4-pinene 34 372 628

2. camphene 46 387 642

3. /3 -pinene 392 691 4. mycrene 72 399 710

5. limonene 112 418 759

7. linalool 151 462 nd

8. methone 224 574 802 8a. naphthalene 600

8b. Unknown aromatic cpd. 604 8c. Unknown C12H16 (?) 688 9. methyl naphthalene 375 705

10. methyl N decanoate 404 736 13. ethyl decanoate 478 nd 17. dodecanol 564 nd

18. /S-ionone 572 858 1024 20. dihydroactinidiolide 883 1260

22. dodecyl acetate 698 1296

26,27. heptadecane 765 1366

Table XX. Peak Identification and Mass Spectra Scan Numbers for Boronia megasticima.

nd = not detected

The total ion current traces of the three samples of

volatiles are shown in Figures XXXI to XXXIII.

A comparison of the relative proportions of the

.components is given in Table XXI.

- 100 -

Peak Component No.

Concrete Concrete Headspace

Flower Headspace

la. Unknown monoterpene. 0.8 0.9 3.5 1. ..f-pinene 1.6 3.1 26 2. camphene 0.5 0.9 26 3. /3-pinene 0.3 0.9 32 4. myrcene 0.5 0.4 3.1 5. limonene 2.6 1.8 3.8 7. linalool 0.3 1.8 - 8. methone 2.4 6.8 0.3 8a. naphthalane 3.1 8b. Unknown aromatic cpd. - 1.3 8c. Unknown C12H16 (?) 3.1 9. methyl naphthalene 2.6 15 10. methyl N decanoate 6.8 26 13. ethyl decanoate 3.4 - 17. dodecanol 9.5 - 18. 4-ionone 21 19 1.8 20. dihydroactinidiolide 2.1 0.9 0.3 22. dodecyl acetate 7.6 - 0.5 26,27. heptadecane 35 - 0.5

Unknowns 3.0 15 2.2

Table XXI. Relative Percentage Composition of the Volatile Components of Boronia megastigma.

ii) Discussion

The relatively high proportion of monoterpene components

in the headspace from the flowers was of considerable

interest, as the aroma from the flowers was considerably

"cleaner" than that of the solvent extracted material.

Leggett [215] reported the presence of a number of

components not normally found in natural Boronia extracts

when he examined a sample of commercial Boronia absolute.

The addition of the compounds, menthol, piperitone, /3-

ionone, menthyl propionate and elemol; may be an attempt by

commercial interests to correct this difference in odour

between the extract and the flowers.

-101 -

Leggett has divided the volatile oils from Boronia

megastigma into a number of different chemotypes on the

basis of the presence, or absence, of certain components.

The headspace from the flowers provided sufficient evidence

to identify the plant as belonging to a particular class on

the basis of the presence of menthone, methyl decanoate,

ethyl decanoate, methyl naphthalene, although the

concentration was low.

A Tenax sample collection tube in conjunction with an

efficient separation and identification system, such as a

GC/MS system has been shown to be an effective method for

the collection and analysis of Boronia volatiles. The system

as outlined could, with a few modifications, be utilised to

determine rapidly the optimum time for harvesting flowers

for commercial purposes. Monitoring the relative

concentrations of the physiochemi,cally active components

over a period of time would provide valuable information for

the commercial grower as to the optimum harvest time under a

variety of weather conditions, without having to harvest

selected areas of a field. The technique should also be

applicable to other essential oil crops, as the steps of

(comminution, extraction, and evaporation would not be

required.

- 102-

CHAPTER VI

COMPOSITION CHANGES OF VOLATILE COMPONENTS

OF PINUS RADIATA.

Introduction

The volatile components of Pinus radiata have been

reported by a number of authors in the past decade generally

using one specific method of sample collection and analysis.

This chapter outlines a number of complementary techniques

that have been used to document the changes in the volatile

components occurring upon injury of a tree. Wide variations

in the composition of the major volatile monoterpenes

between individual trees made comparisons difficult. Trees

were selected that appeared to be similar in initial

volatile composition so that changes occurring could be

documented on two individual trees using the techniques

available.

Bannister [217] compared the volatile components of 189

trees of Pinus radiata in several locations in New Zealand

and California and found a wide variation in the percentage

composition. The percentage of -c-pinene varied from 12.6 to

58 percent. The total of plus/3 -pinene was of the

order of 98 percent. Other indentified components were

camphene, limonene and 4-phellandrene. Previous work by

Mirov [218] had identified many of the other major

constituents of various pines including radiata.

- 103-

Damage to Pinus radiata by the introduced Sirex noctilio

wasp prompted investigations into the volatiles of damaged

trees by Simpson and McQuilkin [219] who noted only minor

changes in composition with time after felling of the tree,

even though the attractiveness changed markedly over the

first three weeks. The authors of this paper trapped the

volatiles from sawn logs, with ends sealed, by cryogenic

traps, and noted the change in response of components by the

antenna of S. noctilio. The individual volatile components

identified were ,c -pinene, camphene,/3-pinene, sabinene,

mycrene, 3-carene, limonene ,/2-phellandrene, x-terpinene,

p-cymene, terpinolene as well as camphor, pinocamphone,

isopinocamphone and trans pinocarveol. Tentatively

identified were fenchone, terpinen-4-ol, thymol methyl

ether, borneol and citronellol.

Work done by Madden [220] showed that the timing and

duration of attack of S. noctilio correlated with the degree

of stress undergone by the host tree. Felling of a tree

induced immediate attack with susceptibility for

approximately 14 days. Girdling was correlated with attack

9 to 12 days later, but susceptibility remained for an

extended period i.e. over one season. It was thought that

debilitation of the tree and the reduction of soluble solids

caused the release of the attractant from the cambiumphloem

region. Injection of mucosecretion by the female wasp

appeared to worsen the initial stress condition and result

in attacks 1)3, more females. Stress induced changes altered

the tissue permeability and increased the rates of

- 104 -

monoterpene and water vapour loss through the bark [221].

Additional work has been undertaken in this project to

confirm this finding and to identify and quantify some of

the terpenes.

Alterations in tree chemistry of attacked and unattacked

P. radiata are also given by Hillis and move [222] who

observed changes in the composition of polyphenols in

sapwood, knotwood and heartwood. Ethylene has been reported

to be released in larger quantities and to correlate with

polyphenol production of an injured tree [223].

Sampling and Analytical Methods

The Pinus radiata trees used for the experimental work

were situated in a forest at Pittwater, Tasmania. The group

of trees selected was a closely planted (1-1.5m) trial plot

15 years old.

Mass spectral information, together with relative

retention times on two different capillary gas

chromatography columns, was used to identify most of the

components in the volatile oil and headspace gas samples.

Comparison of the information with literature values and

with authentic standards served to confirm the identity of

all but a few of the components isolated. OV101 and

Carbowax 20M were the two liquid phases used in the 50M

silica GC capillary columns. Conditions of operation are

set out in Table XXII.

- 105-

Gas Chromotography

Column Type carrier flow (ml/min) carrier gas

OV101 Scot (silica) 1.0 hydrogen

CW2OM Scot (glass) 1.5 hydrogen

Initial Temp. (deg.C) 50 50 Final Temp. (deg.C) 200 200 Temp. Prog. Rate (deg.C/min) 4 4 Injector Temp. (deg.C) 240 240 Detector Temp. (deg.C) 250 250 Inj. Vol. (microliters) 0.05 0.05 Detector FID FID

Gas Chromatography/Mass Spectrometry

GC Column OV101 (silica) Interface Temp. (deg.C) 250 Mode Electron Impact 70 Ev Scan Rate 1 second per decade Mass Range 20-350

Table XXII. GC and GC/MS Operating Conditions.

- 106-

A. The Composition of the Steam Volatile Components From the Bark Oil of P. radiata.

Earlier work by Simpson and McQuilkin [224] had

established the occurrence of fifty components in the steam

distillate of the bark oil. However, it was felt necessary

to document these components using the GC and GC/MS

facilities presently available. A section of the bark-

phloem was carefully removed from a number of trees and 500g

was broken up into small pieces and steam distilled. The

yield was 0.3 percent.

High resolution gas chromatography using the OV101 and

CW2OM silica capillary columns indicated the presence Of the

two major peaks,qt/. and pinene, and a number of smaller

component peaks.

The flame ionisation detector trace for OV101 is shown

in Figure XXXIV. The GC/MS total ion current trace for the

equivalent CW2OM trace is shown in Figure XXXV. Scan

numbers and peak identity information is shown in Table

XXIII. A number of minor components were not identified but

mass spectral information and the relative retention index

was determined for each component. Elution order of some

components did not always agree with the literature values

[212] but a slightly higher programming rate and changes in

the retention properties, (especially of the CW20M), could

account for the discrepancies.

- 107 -

Identity Composition Scan No.

Ret. Index

Base Peak

Parent Peak

a-pinene 32.8 154 1023 93 136

camphene 0.5 176 1075 93 136

B-pinene 55.2 205 1120 93 136

pinane ? , 0.1 226 1140 95 138

A3 carene 0.9 231 1145 93 136

myrcene 1.8 240 1150 93 136

cymene (type) 0.1 249 1175 119

limonene 2.4 257 1195 68 • 136

dodecane 0.1 271 1200 57 170

B phellandrene 0.4 273 1215 93 136

unknown 0.1 278 1220 71 154

m cymene <0.1 313 1265 119 134

p cymene 0.2 321 1270 119 134

a terpinolene 0.1 329 1285 121 136

C13

hydrocarbon + unknown <0.1 349 1300 57 184

myrtenol <0.1 371 1335 79 152

unknown mixt 0.1 396 1365 97 154

cymene (type) <0.1 403 1375 119 134

unknown 0.2 416 1390 67 152

bornyl acetate 0.1 430 1405 95 158

fenchone 0.1 438 1410 81 152

1450 a-dimethylstyrene 0.1 466 *1278 132 1 .32

unknown sesquiterpene <0.1 495 1485 119 204

a copaene <0.1 518 1520 161 204

dimethylstyrene plus unknown <0.1 523 1525 117 204

iso pinocamphone 0.2 549 1545 83 152

pinocamphone 0.1 575 1580 83 152

1585 linalool 0.1 579 *1506 71 154

unknown sesquiterpene <0.1 588 1595 161 204

1605 iso borneol <0.1 601 *1660 95 -

1015 fenchol 0.4 608 *1575 81 204

6 tert. butyl cresol <0.1 614 1620 149 164

terpinene-4-ol 0.1 628 . 1630 71 154

cont...

- 108-

myrtenal .+0.1 651 1660 79 150

transpinocarveol 0.8 678 1690 no spectra printed

borneal plus unknown 0.1 683 1700 95

1728 a terpineol 0.6 714 *1661 59 not present

muurolene <0.1 728 1730 105 . 204

A cadinene <0.1 755 1760 161 204

unknown 0.3 795 1810 79 152

calamenene <0.1 819 1840 159 204

p cymene-8-ol 0.1 840 1850 135 . 150

* represents literature values.

Table XXIII. Mass Spectral and Retention Index Identification Data for Whole Bark Oil Distillate P. radiata. CW2OM Capillary Col. 50-200 deg. C. 4 deg./min.

Table XIII lists over forty components in the steam

distillate of the bark oil from P. radiata. Twenty or so

other components were noted in varying trace concentrations.

Many of these were not adequately resolved on either the

OV101 or CW2OM columns, and rearrangements to isomers would

probably occur on separation. This work has substantially

confirmed the results of Simpson and McQuilkin [224].

29

2 I

4 6

- 109-

auau!d -.3

---'

.

0 c U c

1 ,r

0 C

<

—

4,

• .7

L

0 > , m U 0 C _ a o c E 4,

-

u

,

•

L 20

30

40

50 60 70 Time (mins) 80

Figure XXXIV. GC Trace Bark Oil P. radiata OV101 Capillary Col. 50-200 deg. C at 2 deg./min.

CRL:IC3S0

222 112:22

17:11

85557

7 . 7

7S

0

7E2 WM

123 24.2 20:53

27:44

vos

az. 1116 2:22

9

651

-

l v 2

Figure XXXV. TIC Trace Bark Oil P. radiata CW2OM Capillary Col. 50-200 deg. C at 4 deg./min. Peak Identification Table XXII.

- 110-

B. Collection of Headspace Samples From The Bark of P. radiata.

Collection and trapping of the volatile components

emitted from the bark were undertaken by placing alpiAythene

sheet collection bag round the tree and drawing the

headspace through a Tenax sample tube to collect the

components.

Each trap consisted of three pieces of 40mm wide

aluminium strip bent so that the upper and lower ends rested

against the trunk and the centre section stood away 100mm

thus providing an annular space to collect the volatiles.

q0 0mm

Figure XXXVI. Aluminium Framework for Headspace Trap.

The three aluminium strips were placed round the tree at

120 degree intervals and polythene film was wrapped round

the outside and held in position by rubber straps and a

bolted cover strip over one of the aluminium supports.

Large 16mm dia. bolts with 4mm centre tapped holes, held

the cover strip in position and at the same time allowed

access to the headspace inside the polythene. The centre

hole was plugged by a small threaded brass bolt before and

after sampling.

Placement of the traps round the tree was undertaken 24

hours before sampling of the headspace commended. Headspace

sampling was undertaken by attaching a 170mm long glass tube

filled with previously conditioned Tenax GC to the 16mm dia.

bolt with a suitable length of Teflon tubing. The centre

4mm bolt was removed and the glass tube butted up to the

hole through to the airspace between the ;polythene sheet and

the trunk. Air was drawn through the Tenax at a constant

flow rate by regulating a needle valve in the gas stream

before the air pump.

Prior to the placement of polythene sheet headspace

traps round the tree, a sample of headspace from the sheet

was taken by placing it in a previously cleaned large glass

vessel and trapping the components given off when the vessel

was warmed. The glass vessel was cleaned by heating to 600

deg.0 and then allowed to cool. Polythene sheeting was

placed loosely in the vessel and the volatiles adsorbed by

withdrawing air from the vessel through a Tenax trap. Dry

high purity nitrogen was admitted to the vessel via another

Tenax tube. This blank run was made in order to

characterise any components that may have resulted in using

polythene as a containing medium for plant volatile

headspace traps.

Considerable quantities of volatile material were

emitted by the polythene sheet (Figure XXXVII) and the mass

spectra and retention times for the major peaks were

recorded for future reference.

- 112-

14E16

792

7 5 1 2

468

9 3

663 73S 2

868

4'S

.7Q4

3 9

23

zsea

SOO C.S.9 CO9 C.11

13313 ze 315 27 ■ 61

Figure XXXVII. TIC Trace of Sheet Polyethylene Volatiles. OV101 Capillary Col. 50-200 deg. C at 4 deg./min.

1269 31. 9

*at £1

- 113-

C. A Comparison of Bark Volatiles From a Girdled P. radiata.

Examination of the volatiles emitted from the bark above and

below the girdle of a standing Pinus radiata were made to

document the changes that occurred when the tree was placed

under stress. Samples of the headspace and bark oil were

collected and a moisture trap was used to determine the

transpiration difference between the bark above and below

the girdled section of the trunk.

The tree selected was 15 years old and 160mm in diameter at the

girdling point. A 50mm wide ring of bark was removed from the

trunk 2m from the ground and the headspace traps were placed

round the trunk immediately above and below. A 14 day time

interval was allowed-between girdling the tree and the

sampling of the headspace as this marks . the initial stage

of attractiveness for S. noctilio.

Headspace from the trunk above and below the girdle of a Pinus

radiata tree was examined by adsorbing the volatiles on a

Tenax sample tube and GC and GC/MS techniques were used

to identify the components.

The samples were collected in early March 1981 just

before the end of a relatively dry summer period. A previously

dried and weighed copper sulphate tube placed in series with

the Tenax trap allowed the water transpiration of the two

sections of the trunk to be calculated. Air was withdrawn at a

rate of 41mL/min for two hours i.e. a total volume of 4.92L.

-114-

The volume of headspace round the trunk enclosed by the polythene

sheet was calculated to be 9.99L. The copper sulphate tube was

in series for 1 hour and therefore the volume of moist air was

2.46L in each case. Moisture adsorption above girdle was

0.0096g and below girdle was 0.0137g, or expressed in terms

of area of bark 12.5 and 21.6 mg. m -3 . h-/ . These figures are

in agreement with the findings of Madden [221] although a

slightly different technique was used to measure the rate

of water loss.

Following the removal of the headspace trapping framework from

the tree, sections of bark from above and below the girdle were

removed for distillation of the steam volatile oil. A comparison

of the composition of the two oils may provide an indication of

changes that occur in conjunction with the volatiles released

through the bark.

Traces of the GC run on the two samples of distilled bark ,

oil are shown in Figures XXXVIII and XXXIX. The runs were conducted at

relatively high sensitivity (full scale deflection representing

0.2 percent of the mixture), to highlight the differences in the

oil. Identification of a number of the peaks was not possible

due to very small quantities present and the inability to

separate sufficient quantities for confirmatory MS or infrared

spectra. Peak numbers and where possible identification

information are set out in the following table (Table XXIV).

15

Figure XXXVIII. GC Trace of Bark Oil P. radiafa Below Girdle. OV101 Capillary Col. 50-200 deg. C. at 2deg./min.

4 3 5

30

19

1,v11 A.44,,J101 V4AAJ)

2

13

12

26' 36

—115—

24

I 30 ,

13

0 35

3 ' 3

-7 II 12 29

1 I 15 22

26

PI .

14

qkrLI 1\ kA9

I 17 :ic,,9

1 , A 0

/\-\,/■"&ik\i^iv\i' ''')

. d

i\l u- I RI ;

Figure XXXIX. GC Trace of Bark Oil P. radiata Above Girdle. OV101 Capillary Col. 50-200 deg. C. at 2 deg./min.

FID

Res

p ons

e-3

1124

334

4

167

20

16

jt a 1 Da Zica 380 0.0 2.23 6141 lase

8223 868 16140 20110

1 729 Begi 2.3132 26132 33:17

421

422 13327

733739 G .F . =2

—54 8

60

S'S

4 4

2 9

--1--- 208 le:3

f ------1— ota 133e

i i ' £90 16:53

i - - 4-----r---T---T---e 600 702 202 Z023 23:St 27:13

1-- 322 32.25:

1,—. A - r

glee ems 3325

--T-- , V32 Gel7

5 s

81

671

- 116-

Figure XL. TIC Trace of Bark Oil P. radiata Below Girdle. OV101 Capillary Col. 50-200 deg. C. at 4 deg./min.

Figure XLI. TIC Trace of Bark Oil P. radiata Above Girdle. OV101 Capillary Col. 50-200 deg. C. at 4 deg./min.

- 117-

Peak Identify No.

1 tricyclene ? 2 -e-pinene 3 /3-pinene 4 pinane 5 myrcene 6 73phellandrene 7 limonene 8 ? 9 ? 10 ? 11 fenchone 12 ? 13 fenchol 14 ? 15 ? 16 ? 17 ? 18 ? 19 transpinocarveol 20 ? 21 borneol 22 ? 23 terpinene-4-ol 24 °4 terpineol 25 myrtenol 26 verbenone 27 ? 28 citronellol 29 mixture 30 ? 31 ? 32 thymol (or carvecrol) 33 ? 34 ? 35 ? 36 ? 37 ? Other

Rel.Ret. Time

0.974 1.000 1.056 1.077 1.090 1.103 1.114 1.132 1.146 1.159 1.177

Percentage Composition Below Above - Girdle Girdle

4.8 3.0

28.9 16.1

57.8 66.8

0.3 -

1.2 1.2

0.7 '<0.1

1.3 2.3

1.1 0.7

0.1 <0.1-

<0.1 -.(0.1 -<0.1

1.188 .D .4;0.1 0.1

1.219 0.2 0.2 1.325 (- 0.1 0.1 1.627 0.1 0.2 1.772 ,< 0.1 '.<0.1 1.786 cO.1 0.3 1.900

,-, <0.1 ')<0.1

1.922 0.1 0.3 1.963 <0.1 0.1 1.976 0.1 0.1 1.988 <0.1 0.1 2.031 <0.1 0.1 2.050 0.6 1.8 2.067 -4:0.1 0.1 2.081 %0.1 0.3 2.107 \0.1 0.3 2.115 %0.1 0.6 2.139 0.3 1.1 2.169 0.3 0.4 2.219 -.(0.1 0.3 2.227 0.2 1.2 2.238 -.(0.1 0.5 2.249 -40.1 <0.1 2,264 0.2 1.1 2.317 -<0.1 0.6 2.344 0.1 0.4

0.3 0.4

Table XXIV. Peak Numbers and Percentage Composition of Steam Distillate of Bark Oil Below and Above Girdle of Pinus radiata.

The bark oil below the girdle contained a larger

proportion of the more volatile components but generally,

the two oils were very similar in the oxygenated and

sesqui-terpene regions of the trace. The oil below the

-118-

girdle contained a slightly larger quantity of solvent hence

the relative difference in peak area ratios in the traces.

Quantitative interpretation of the results on the basis

of the one set of samples is suspect and further samples

would be required to confirm any differences such as the

apparent increase in relative concentrations of the

oxygenated sesqui-terpenes eluting towards the end of the

chromatogram in Figure XXXIX.

Comparison of the GC traces of Figures XXXVII and XXXIX

with the corresponding GC/MS traces (Figures XL and XLI)

reveals loss of the and /3-pinene and early monoterpene

components, and a massive increase in the relative

proportion of oc terpineol. This may be aue to the use of a

splitter in the inlet of the GC/MS unit reducing the amount

of volatile components reaching the inlet of the GC column,

or to evaporation during sample transfer operations. The

initial percentage composition figures shown in Table XXIV

represent an accurate reproduceable comparison of the bark

oils as three sampling runs showed only small variations.

Ten microliters of Freon (dichloro difluoromethane) was

injected into the headspace, prior to sampling, to act as an

internal standard. In each case the recovery of the added

solvent was within acceptable limits (90 percent for

headspace components above the girdle and, 86 percent for

headspace components below the girdle).

- 119 -

I 2847

5

8

2:ZG caa 17,2E

es, 64-- o: 12 ,r7 6:S7 10:CZ

722 OOP 522 C0.27 C7IZ C7:C7 22:SI

Figure XLII. TIC Trace Headspace P. radiata Below Girdle. OV101 Capillary Col. 50-160 deg. C. at 4 deg./min.

76

1 IGO 2443 290 CA2 COO 7aa 2:ZI Gl1C i40:0 13120 IS:SC COI12 Z2:3S EGaSC

102 Z20 222

Figure XLIII. TIC Trace Headspace P. radiata Above Girdle. OV101 Capillary Col. 50-160 deg. C. at 4 deg./min.

- 120-

The volatile headspace TIC trace for components trapped _

from the bark below and above the girdle are shown in

Figures XLII and XLIII. The traces show very few

differences in the qualitative composition of the volatiles.

Each sample was collected under identical conditions (2

hours sampling time at the same flow rate of 'dry' air), and

quantitative measurements show that the amount of volatiles

released from the bark above the girdle was greater than the

amount released from below. This result in apparent

contradiction with the composition of the oil distilled from

the bark as shown in Table XXIV. Table XXV details the

quantitative information determined from GC runs on

duplicate headspace samples and related back to the MS

traces.

H/S Below Girdle Scan No. % Comp.

H/S Above Girdle Scan No. % Comp.

198 0.1 220 0.8 307 29.4 229 27.3 317 1.0 235 2.9 352 68.0 261 58.0 nd 279 0.6

398 1.1 293 2.1 0.4 8.3

Component Identity

tricyclene ? .,c-pinene camphene /3-pinene myrcene limonene Other

Table XXV. Percentage Composition and Component Identification P.radiata Headspace Above and Below Girdle. nd - not detected

The trace components present in the headspace were

difficult to identify due to the large proportions of

and /3 -pinene. A greater number of components were present

in the headspace from above the girdle, however many were

present at a level below the detection limit of the GC/MS.

- 121-

Attack by S.noctilio occurs in greater numbers below the

girdled or injured section of a tree [220], and therefore,

the attractive component, if such a component exists may be

present at a very low concentration. Alternatively, the

presence of oxygenated components may act as a repellent to

the insect and reduce the incidence of attack above the

girdled section of the tree [225]. Headspace sampling using

porous polymer trapping techniques may not provide

sufficient capacity to trap low concentration components

(40.1%) in the presence of massive quantities of other

components.

- 122-

D. Composition Changes of Bark Headspace of P. radiata.

•

Changes in the volatile components emitted from the bark

of a number of trees were examined using a gas tight syringe

and direct injection onto a packed GC column. Wide

variations in the quantitative composition of monterpene

components of P.radiata have been reported [217], and bark

samples were collected from 7 trees of similar age and

appearance to determine the range of the two major

components.

A 50mm wide section of bark was removed from round the

circumference of each tree and placed in a glass jar fitted

with a lid containing a rubber septum. The containers were

immediately taken to the laboratory and heated to 40 deg.0

and 4mL of the headspace was injected onto a 2m CW2OM packed

GC column. The FID traces of the 7 samples recorded at an

isothermal column temperature of 60 deg.0 are presented in

Figure XLIV. Peak identities and concentrations are

recorded in Table XXVI.

After 6 and 14 days, aditional headspace samples were

taken for analysis using,the same method as outlined above.

The traces recorded on the sixth -day were similar to the

ones illustrated in Figure XLIVa however marked changes had

occured by day fourteen. Rearrangement of .C-pinene to

tricyclene had occured with many of the bark samples in the

closed glass container [226],( Figure XLV).

XI

13- er ii leo XII

1. - terpinetA

II

IX X

P - Cp1ICIIC -terpincol

OH HO

•H 2 0 H'' OW"

'OH

--- II III IV V VI

/1-pinctie rattiplultic tricyclenc terpitiolenc (lipctitenv

OH OH .

VIII -y-terpinenc

XV XVI XVII XVIII ecp 10- em lo- borneoI isobonIcol

fenclmI

nt-pincne

L r VII

a-terpincne

XIV IA-cincole

OH

- 123-

Figure XLV. Terpene Products Resulting From Hydration and Isomerisatibn ofoe-pinene [226].

b)

a)

I I :

;

1

Figure XLIV. P. radiata Bark Headspace. 4mL direct injection on CW2OM packed col. a) Day ,O. b) Day 14.

- 125 -

Tree Number Component 1 2 3 4 5 6 7

1. 01.-p1nene 42.3 36.1 24.5 26.3 34.1 23.7 31.4 2. camphene 3.4 3.8 3.3 4.9 3.9 4.1 2.9 3.4-pinene 41.3 38.0 32.5 37.9 39.4 38.6 48.5 4. myrcene 1.8 12.0 13.8 13.9 6.3 12.5 5.7 4a. unident. 0.4 2.1 1.7 5. limonene 8.0 23.4 13.4 11.6 18.6 9.2

Table XXVI. Percentage Composition of Headspace from Samples of P.radiata Bark. CW2OM Packed GC Col.

Trees 5 and 2 selected for the previously described

headspace work, and samples of the headspace were collected

from above and below the girdle by filling a Teflon gas

sample bag. Direct injection of 4mL samples of headspace

onto the packed CW2OM column confirmed the higher rate of

release of monoterpenes from the bark above the girdle

(Table XXVII).

H/S Above Girdle H/S Below Girdle Component Peak Area % Comp. Peak Area % Comp.

Unknown' 1234 4.6 1043 20.7 -c-pinene 10249 38.4 1094 21.8 camphene 632 2.4 1167 23.2 /3-pinene 14565 54.6 1721 34.2

Table XXVII. A Comparison of Peak Areas (arbitary units) and Percentage Composition of Headspace Components Collected by Gas Sample Bag from P.radiata.

The relative error for the headspace sample collected

from below the girdle would be high due to the small peak

heights (Figure XLVI), but serve as an indication of the

proportions of volatiles released.

- 126 -

CHAPTER VII

CONCLUSIONS

Techniques have been described for the sampling and

identification of trace amounts of volatile organic

compounds from natural products and air samples. Tenax has

proved to be a versatile trapping medium for the collection

of a wide range of organic compounds from gases to high

molecular weight terpenes. The equipment used to trap the

volatile components is relatively simple; consisting of a

Tenax sample collection tube, a flowmeter and an air pump;

thus enabling the collection of samples in field situations.

The quantity of material trapped can be controlled by the

volume of air passed through the trap, up to the point where

breakthrough of the most volatile component occurs. This

point is determined by conditions such as the temperature,

flow rate, and chemical nature of the component, and can be

monitored by the use of a second back-up sample tube in

series with the first.

Limitations of the sample collection procedure include

the early breakthrough of some low molecular weight gases,

and the difficulties associated with the detection of trace

quantities of components in the presence of large quantities

of material eluting at the same time. The selection of

other trapping mediums with different polarities and the use

of subambient temperature programing on a GC - would assist in

overcoming some of the above problems.

- 127-

Desorption of trapped components by heating and

secondary trapping within a U tube enables optimium

introduction of the sample onto the GC column using flash

injection techniques. To prevent thermal degradation of the

sample, elution temperatures and times need to be monitored

to ensure adequate recoveries with minimum alteration of

labile components.

The quantitation of components in complex mixtures of

volatile unknowns presents problems. The adsorption and

desorption efficiency of each component will vary according

to the concentration of other components in the mixture and

the chemical nature of the adsorbent, as well as on other

factors such as flow rate and temperature. Tenax provides a

versatile trapping medium for many complex mixtures and

quantitation may be achieved using addition techniques for

components of interest.

Identification of separated components was generally

carried out "on the fly" using GC and GC/MS techniques as

there are considerable difficulties involved in collecting

the microgram to low nanogram quantities of material

separated by the high resolution capillary GC columns, and

performing off line identification. Additional spectral

information was often required to complement the GC

retention time and mass spectral information that was

available. The introduction of a Fourier-transform infrared

system coupled to the GC will assist in the confirmation of

- 128-

the identity of many of the trace components detected in the

samples examined in this work.

- 129-

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•

4 GIANNOVARIO, J.R., Grob, R.L., Rulon, P.W. J. Chromatogr., 121, 285(1976)

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- 143 -

APPENDIX

SECTION A. Paper Published During The Course of This Work.

' SECTION B. Equipment Details. Gas Chromatography Equipment. Gas Chromatography/Mass Spectrometry

Equipment Used During The Course of This Work.

SECTION C. Mass Spectral Information From GC/MS Runs (Arranged According to Chapter)

Chapter IV Tenax Trap Volatiles.

Chapter V Drimys lanceolata Prostanthera lasianthos Bronia megastigma

Chapter VI Pinus radiata

- 144-

- 145 -

Paper in attached pocket - inside rear cover.

- 146-

GAS CHROMATOGRAPHY

• Packed column gas chromatography was performed on

a Varian 3700 gas chromatograph equiped with flame ionisation

detectors, and a temperature programme controlled oven. Two

meter long by 4mm internal diameter glass columns were used

with nitrogen carrier gas at a flow rate of 35mL per minute. •

The same GC was used for capillary column gas chromatography

using a SGE capillary column conversion kit to optimise gas flow.

GAS CHROMATOGRAPHY/MASS SPECTROMETRY

A Pye Unicam Series 204 gas chromatograph was coupled to

a VG Micromass 7070F mass spectrometer via a VG jet separator

(for packed column operation), and operated at a vacuurn of

4*10-4 Torr. Capillary columns were coupled directly to the ion

source. The system was operated at 70 EV and data was stored

and processed on a VG 2235 system coupled to a PDP 8/A626

minicomputer. The electron impact (ei) mode was used on all

GC/MS runs. Calibration of the system was performed by

injection of fluorinated kerosene mixtures. The carrier

used was either hydrogen or helium at a flow rate of

1 -1.5mL per minute.

)00 CADOOS 24 TENFIx TRAP VOLS CFR. : ICSOS

4:4

9056

GS

• 1 - gam 1

2S—JUN-81 220

• II 1

acetone

022s3

147 -

COMPONENT MASS SPECTRA

CADOOS 20 TENAX TRAP vOLS 2S—JUN-81 [Pa.: cSoo Hydrocarbon (pentadiene)

I CO

200

CROOOS 28 TENAX TRAP vOLS 2S—JUN-31 CR.:IC:SOO methylene chloride + acetone + unknown

43 25217

_

- CD

100 CANS 29 TENRx TRAP vOLS

Icsoo methylene chloride 4'1

200

;!ro :fito

CSilS

300

2S—JUN-21

; a.

a.

- 148 -

CROOOS 32 TENAX TRAP VOLS ca.:1MM Freon

2S-Jur\I-81

39112

IS

100 209

320

CFIDOOS SO TENAX TRAP VOLS

2S-JUN-8I CIA.: 1(W2

carbon dioxide

300

CADOOS 70 TENAX TRAP VOLS 2S-JUN-n-pentane + methyl pentane CFtl. : 1 CS 90

13972

300

2S-JUN-8 log zoo

CADOOS 75 TENnX TRAP VOLS ca.: csaa chloroform

GS2-7S

7— "7"-*- 2.0

benzene 25-JUN-81 CADOOS 114 TEJA 1Rcp VOLS

C: lases

13152

79

- 149 -

ee 200 S'01,7

CADOOS 119 TENAX TRAP VOLS C.RL : 1 Cat/ chloroform

'2S-JUN-81

31 3S

0

-

47

- -r r -ZOO 13211 ef.,1

CADS 245 TENRX TRAP VOLS 25-JUN-21 CAL:ICSE2 toluene

)00 CF1000S 302 T ENAX TRAP VOLS CRL:ICSSO

r ZOO

tetrachl oroethyl ene 300

25-JUN-81

1472

1. 1 1 ii

I 4 • 1 I II

- Lr

44 1Nfr;

•44

5,G38

phenol 2S-JUN--81 CROOOS 432 TENAX TRAP VOLS .

cm.:Icsso 94

3220

phenol 2S-JUN-81

lea CR0005 438 TENAX TRAP VOLS ca.: 1C:560

r 300

i 77 i

I I l i 1 , l it) , lilii, , III, 1, • III

I VA!

- 150 -

a

r-- -f Z00 2vo

25-Jurl-e leo

CRDOOS 469 TENAX TRAP V OL.S CAL:ICS:00

cresol

a

CADOOS 459 TENAX TRAP VOLS CR1. : CS0.0

128

25-JUN--B cresol

1 ;-? G

3294

3058

- 151 -

CADS 490 TENAY TRFP voLS ethyl phenol (?)

,

77 i! I !I

11

I

C I 4..1(111

fa S

CAL: 3 cses

CHDOOS ciSS TENFix vOLS GL: IM2.0

•

IF NH •

dimethyl phenol (?)

1:02

2S-JUN--8 2

I 07

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CADOOS 502 TENAx" cRL:Icsea 2 ethyl phenol (?)

2S----JUN---

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;co Pi2I

CADOOS 61 TENt,Cx cRL:3csen pentadecane

7115

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■

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9552

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6f3

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- 152 -

CADS 642 TENFIX TRW VOLS

2S—JUN--81 CRL:V=S22

Unknown I 4528

i 1 ; . ■ 1

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2S-JU-9 I

C2

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667

b1/4 /!ii 13"1.1 I Pi i 1

a ea

200

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127 OR I mYS L. CW20M SILICA 80-200 4

4084

41

0E0001 cAL:ID400

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300

26—AUG-82 4,0

4.1 947

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1 '300

- 156 -

0E0001 96 ORIMYS L. CW20M SILICA 80-200 4 RUN 2 CAL:1D402 STA:E.

SOLVENT (CHLOROFORM) 26—RUG-82

3:1

100 200

0E0001 96 ORIMYS L. CI-120M SILICA 80-200 4 RUN 2 CAL:1G402 STR:E.

300

26—AUG-82 3:5

7 487

I ,

100

200

300

DE0001 116 DRIMYS L. CW20M SILICA 80-200 4 RUN 2 cRL:ID488 STA:E.

//3 -PINENE

26—RUG-82 3.39

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CAL:18400 MONOTERPENE ( ?)

P CYMENE

100 200

0E0001 170 DRImYS L. CW20M SILICA 80-200 4 RUN 2 CAL:18460

^-1 P• l.r. ,11!

182 200 300

6:25 CAL:18408 UNKNOWN

-158-

619

300

26—AUG-82 S:21

0E0001 206 ORImYS L. CW20M SILICA 60-200 4 RUN 2 26-AUG -82

2

I -

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I SOMER OF X

S7

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100 ?.00 260

DE0001 1S8 ORIMYS L. Cw20m SILICA 80-200 4 RUN

300

26—FiUG-82 7:3

2824

26—RUG-82 4:se

2827

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Ips . 1p

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.3kto 26—ALIG-82

8:46

811

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200 • 306 _

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CV

2

1 1

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- 159 -

CL-42{irl SILICA 80-20 4 RUN 2

ISOMER OF X 26—AU0-22

8:2S

879

26—AUG -82 933

DE0001 304 ORIMYS L. CW20M SILICA 80-200 4 RUN 2 caL:Ipaela STA:E UNKNOWN

207

i

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, • { i 1 .--' i S;S

II

: i t l ,l 1 1H I 1 i

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DF0001 32S DR' MYS CAL1D'r00 Sla:E.

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-BOURBONENE

300

26—ALIG—P2. 1o:13

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100 . 200 DFOC:_101 31-2 idRIMYS L. CA-120r: Si Li CA 80-20(?; 4 RUN 2

UNKNOWN ( X + CH2 ) _ • --- CAL: 1D400

3co 26—RUG-82

10:4S

2349

- 160 -

DEEIOH1 332 Mims IJ. e',..i22t1 SILICA 60-202 4 RUN 2 0: CAL: 1040.0 STA:E.

UNKNOWN 126

_ 112

I

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L - 1 : e3

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CAL: 1D4 STA:E. L I NALOOL

•

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ZOO 300 4 RUN 2 2 .6 —AUG 2

128 CAR VU PHY LL EN E

493

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DE.0001 4S5 CR NYS L C:-/20r1 1L. CA S0,-20 4 RUN 2 CAL: 1D400 STA:E. MONOTERPENE ALCOHOL

- 161 -

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TERPINENE-4-01

12.25

756

• I

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DE000] 42S P,R mYS L. C1-12Cir1 :1;11.. 1. CP. RUN 2 CAL: 111460 STA:E. SESQUITERPENE

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14:S7

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3NO1I213dId 81, :Si • 3 :YIS 00 b01 C1E13

Z8-9nu-SZ . z NnH i, gez-es 03I1IS Weqt13 '1 SAt-HUO EfaS 100 00 :

......—.1....._.....-- i.........- ..... .1 ,::::1 -.....J .....zt.1—.....-,-.. 1 _T I

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Nnu eoz-os U311IS - 1 SAW 180 18S 100030

— E91 —

- 164 -

DE0001 738 DRImYS L. 0,420m SILICA 80-200 4 RUN 2 CRL:10408 STA:E. METH.. EUGENOL (TYPE)

26-AUG-82 23:11

431

57

5, 71

9 1 147

163

t 14111 py, 1111 1, 1 k 1 11! 1 1 ii11 1 11 Pi Ic 1 4 ) I I

100 '200 DE0001 771 DRImYS L. CL42011 SILICA 80-200 4 RUN 2 CAL:10400 STA:E. SESQUITERPENE ALCOHOL

300

26-AUG-82 24:13

591

41

57

204

9

161

27

5,

. 41,41(11

81

1,1,1 7.1

J1 1111 11 100 200 • 300

0E0001 779 DRIMYS L. C1420M SILICA 80-200 4 RUN 2 CRL:10408 STA:E. SESQUITERPENE ALCOHOL

26-AUG-82 24:28

603

105

100 200 DE0001 801 DRImYS L. C1-J20m SILICA 80-200 4 RUN 2 CAL: 10400 STA: E• GUAIOL

300 26-AUG-82

25:10

161

4095

4

4

57

1 3

161

1 7 9 204

11[

121 1 7 189.

41

204

1,

8

7

208 380

DE0001 826 ORImYS L. .CW2OM SILICA 80-200 4 RUN 2 mu1l-3400 STR:C. OXYGENATED SESQUITERP.

26-AUG-82 2S:S7

— 165—

01 k

619

3

GB 91

105

SS 27 133

LA)116 114 11i,I,Ay4 1. ,... 1,4,141! L 114,ji IA, I ,(1 100 200

ORImYS L. Cw20m SILICA 80-200 4 RUN 2 STA:E. MGENOL

205 153

1 46

1 97 20

0E0801 88 CAL: 13402

, 300

26-AUG-82 2G:S7

1900

•

.4

143

77 31

131

108

200 300

.s;

DE0001 861 CRImYS L. Cw2OM SILICA 80-200 4 RUN 2 CAL: 1[3.4043 STA:E. ISOMERIC GUAIOL (?)

26-AUG-82 27:3

I 9 204 GSG

-

133

41 0 105

Lii i .1 iIi 1 11

1-9

,k,. , .,.1.„ . , L. , 20

DE0001 874 ORImYS L. CI-12@M SILICA 80-200 4 RUN 2

26-AUG-82 CAL: 113400 STA:E. UNKNOWN 27:27

2

121

1-1

225

71 9) S7

204 09

311

, Ji i 1 I 11 111 1/1 1; 111 1J id il ilt itilli II Jil Ilh LIJI IL ■ 111 Tilt , ILJ b I. 1 .. 1 100 200 00

DE0331 909 DRIMYS L. C1-120m SILICA 80-200 4 RUN 2 CFit_: 113400 STA:E. UNKNOWN

26—AUG-82 2026

121

161

IS 193 1 7 4 09

fr( iJb 41,11 4 100

204

300 ti. .

200

- 166 -

0E0031 885 DRImYS L. C1420M SILICA 80-2ils 4 RUN 2 CAL: 10400 STR : E. METHYL EUGENOL + CH2 !GI

26—F1UG-82 27,48

q 71 172

3 1 1 9

7 10S

91 GE 1'1 3

I S9

d1,1 ki 1, Ili 1 11 I WI

ii

ill 1 idli

1

I, 11 11 II

9

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62 202

ti 111,, 111011,11 I 1 11 11 i A , A I IA 300

26—FIUG-82 283S

ro

to 7

I 0 0 0 61. •••• • fk

100 200

0E0001 910 DRImYS L. CW20m SILICA 80-200 4 RUN 2 MIL:10400 STA:E.

0,q UNKNOWN

4J

17

m-

w

CD

71

S7

Le, ii

27

10E03

4

41 28

7 1

200 • 300 188

- 167 -

26—RUG-82 29:11

• DE0001 929 DRImYS L. CW20m SI L I CR 89-200 4 RUN 2 CAL:11)408 STA:E. MYRISTICINE (?)

192

91

6 13 1SS

1 Alps 41 11 , ktbe1l4 . 11 111 lc )4..1.4 .. I els , , r.

100 288 DE0001 101S DR I mYS L. CW20m SILICA 80-209 4 RUN 2 CAL: 1D400 STA:E. UNKNOWN

1623

1300 26—AUG-82

31:63

IC

2184

03—DEC—PW 10:c

302 - 03—DEC-80

le.ss

200

DEBRO4

/ "t

41

- 168 -

MASS SPECTRA

574 .90RONIF: FLOWER VCIL.RT

MENTHONE Peak 8)

1:43 1 /1

1$4

1.1 0 1, 1 ' Igo

BORONIR FLOWER VOLF1TILES

NAPHTHALENE (Peak 8a)

e 1

1 11

Iclat

DEf3R04 600 CRL : 1C302

19S3

03—DEC-80 Unknown Aromatic cpd (Peak 8b) 19.2

651

I,

2 Oil

;OH

03—DEC--;22. Unknown compound 19:Z7

Zoo

d fri, ■

44

I. 1 6,'. g...H ._,.,. L „,ij

I , , .!1,

i . . . .1,

23S1-

102

132

300

DEFiR04 624 BORDNIR FLowER

_

31 r. 1

•

..

4,1

I 0 1.1 1.11 I :1 1 1 11 1 .., : 1.1„h,

122 DEE,F\0 41 6:17 E-313RONIA FLOWER VOLA1 ILES

. ChL:1C320

--219 —4(3

1

1

j0 711 32i

tt, JJ 14 I 1111, d ,1, 14 P :rrt r 320

DEBRO4 G8B ri I Y T LIRE: CRL: 1C300

MASS SPECTRA 03—DEC—RP

MIXTURE TETRAHYDRCNAPHTHALENE AND A D1METHYLINDANE CPO. (Peak 80 21:41

-

100 '200 302

- 169 -

„r METHYL - NAPHTHALENE (Peak 9)

1200 322

0EBR0 736 BORONIA FLOWER VOLATILES c5L:]casa

METHYL N DECANOATE (Peak 10) 23.12

100

DEBF:04 750 DORON I R FLOWER VOLAT I LES cp.L:Icaoa

UNKNOrIN

411

122

-

69

T.;

1 7.00 .;00

03-DEC- Zj.217,

16382

SS0

11328

1216

1 IN

DEBR04 70S cRL:Inera

1 47

111

4! 1! rj I. VA

100

BORON! A FLOWER

1 320

03-DEC,-(3 zz:13

' F

VOLH1 I LF.S

- 170 —

MASS SPECTRA

: •

MF_NTFONE (Peak 8)

! H! 1

7.4 ! 1 I 1 :".,•,: l

'. 1 I It :• I I

il :•,',1 I

.- 11 1 1 III 'II r

' 4I1 II . 1 .111 A. C.: II, 1 . , 1 4 1.

—1 ''''. , i r T , T•

Goa BOROW1H FLOWER VOLRII.LE 03 — DEC--&i. 1

NAPHTHALENE (Peak 8a)

C'Ftc c7

:fL :

-.- i ii 1 : H t

• 1 . i t i t H ...

I 1

DEBRO4 CP.L:Ic3so

-

,

-

,

,

- I I i

P.J1 1...b..r1,4,46-1..g..wiJ........I.J1 !.''' ..._—_, , .g.,.._,. ............i...._. ...!. t 1 —T r -r--------------r

'1. aa Z.'i'l 7.:63CI

D F2 ' G3 cr S2F,.[Its: CrIL::C1420

• : `• 2.• I I 1 i

i 1 1 Z " 11

2 1 1 I

1 1 I I 1 1

,-,•; • 1 ! I 1

-,

.-.=1 J 1 , , , ! I i .: .gg... ,.., .g ..,.....„:„...,,,a,,,,...„..,,,.. i...... f I i II.II J.I.I i .,.. ,),1_,k, 1; gl

- - 'g

9.2 Unknown Aromatic cpd (Peak 8h)

L, • 222

L.:c_n;--•,b4 617 FOR0r:IR FLOWER, voLAULEI.:; D E C ChL:ICa,08

Unknown compound

I !!

li '.... ...

Efi

/H

I P! ; • I ; ! I I .1

! 1 II 41 • ! r • 'T -I -4

.

:7:3 ZOO

617 BORON' A FLOWER VOLAT ILES

141 1. 111 ;

r

GEER04 S7c; CRL:Ic2no

41

f's5

1

— 171 —

MASS SPECTRA

EOF,r, ■.! Ft FL-OWE:F-3 %/CV Lt.::

PCJITHONE (Peak 8) a _

382

03—DEC-80 le.s5

2181

. I AJill 1

188

DEBR04 600 BORONIA FLOWER VOLATILES CRL : 1C389

NAWTHALIFTE (Peak 8a)

03—DEC—P0 1R:C

83

ii

41

, Ike/ [! 11..11 41, d.,_;to. , ,,.. .i. • L IL I { till

L .A. ,

2351

a .

120 ZOO 300

DEBR04 604 BO-FON:A FLOt4E.R VOLR1 -ILES ChL:Ic322

9 Unknown Aromatic cpd (Peak 8b)

03—DEC-80 1 3

91

1359

280

03—DEC--;20 13:27

1 —T

102 200

Unknown compound

GS1

! 47—

--T 32

ZOO 7100

41

I DEBEi24 7Sc1

L.

2e,4.

2Tir

e N,E .) I --r --r

100

BOFON]R FLOWEli vOLAT ILES

11,11 , j 1. 3, I IIfa I IL, II! ft :2•13

0E5R84 688 mix1ul9E-cRL;Ic20o

- 173 - MASS SPECTRA

MIXTURE TETRAHYDRONAPHTHALENE AND A DIMETHYLINDANE CPD..(Peak 8c) 21:4:

1216 -

1

1 _

I 1

'1 ' I

1!1 I, .1 i, 0

I ,

,, , i I

'1!T I

4 0 . ..i. r— T -1" -----r 1 ---1;

9FBR84 7O 0FiGNIFI. FLOEF VOL.HTILL:-.; caL::=32o

METHYL NAPHTHALENE (Peak 9)

,4

Ii 328

1,1 , 1i ,T.1

'10.2 aeo 300

DEBR84 736 aoRor4: FLowER V.ht ILL; criL:Ic302

METHYL N DECANOATE (Peak 1 0)

03—DEC 23212

1G3130

•

;ov

SSi3

1-70.0

:00 200

100 2:10 32o

220

03-DEC-%i.0 19:27

202

Unknown compound

DEBR (aq CRL:1C390

_1

- 174 —

MASS SPECTRA

S74 50soNIA FLoLIER VflLf-U Li-

MENTI-JNE (Peak 8)

NAF1-fTHALENE (Peak 8a)

03-DEC--) 19:G

0EB1904 GOO BORONIfi FLOWER VOLATILES Cf211_:1C3138

1

44

Z3S1

.1 •,.,I _1 .

4 1

DEBR24 604 6 OFiONIF1 FLOL4ER VOLFil ILES CW— :1C999

Unknown Aromatic cpd (Peak 8h) : 1 4

31 7, 1

ass

.;

1/ 1 .1 I . t i , pc.,1 .. • '•

149

15 4

I

03-DEC-80 13tZ

DEBRect 617 .BOROtil A FLOER VOL_Fri I LES ChL:1C1400

r 3 ? - c.

21 119

cinti, 3.1 1.,11 11 ,4

I .;

1 90

21 84

6S1

h , JI. 200

100 ZOO 30.0

;20 03—DEC—E.

2.00 !oo 0EER24 7S0 EC3P,01 ,1 .19. FLOWER VOLHT ILES

SSD

'30:4

111

1 111 1 I I I iNi,ItOthi 10 1 P 11- 1 100

if , A ITti, A , 1 !1

DEBR04 736 BORONIA FLOWER VOLR121..E1:: CrL: 1C2

_71 METHYL N DECANOATE (Peak 10) .

g3—DEC—e.? 23,12

16300

CIL: 1C3

- 175 -

DEBROg 68E3 nixTURE-cRL:Ic300

VASS SPECTRA 03—DEC-20

MIXTURE TETRAHYDROVAPHTHALENE AND A DIMETKYLINDANE CPO. (Peak 8c) 21:41

171

44 47 1E2 .11

1; [I .1

100

DERRa4 72 ORON2 VOL:IT 1 Li7.

1

1 I

-1

320 03—DEC—FIP

METHYL NAP1-MIALENE (Peak 9)

11 328

03-DEC-80 18:6

DEBR04 S74 BORONIFI FLOWER VOLATILES'

MENTHONE (Peak 8) CRL:1C200

1

2010 200

03-DEC-80 18ASS

F

2184 41

SS 63

'3

1'1 83 97 14

. I

cc,

ive3

DEBR04 600 BORONIA FLOWER VOLATILES CML:1C300

2114

44

444.11,14 _ 100

2351

7 —W

200 200

NAPHTHALENE (Peak 8a 128

MASS SPECTRA

DEBR04 604 BORON' A FLOWER VOLAT ILES CRL:1C300

03-DEC-80 19:2

Unknown Aromatic cpd (Peak 8h)

•

es,

1959

91

41

1 i 1 I

• I .---- .....I.J.-..- -.-.4-,,wir...,

100 0C3

20 DEBRO4 617 BORONIR FLOWER VOLATILES

03-DEC-BO

CHL:1C300 Unknown compound 13:27

1 kNr

1 -1

1 1

11

,

1 a 1 0

lb 1111 01111

71

ift

91

ti k L Li 1 • 8

till..

6S1

100

200 00

DEBR04 688 MIXTURE CHL:1C3130

03-DEC-80 MIXTURE TEIRAHYDRONAPHTHALENE AND A DIMETHYLINDANE CPD. Peak 8c) 21:q1

100 12130 -T- 300

MASS SPECTRA

i1

ii

S4 1

,

1

118 131

10

1216

1 11328

100 200 202

DEBR04 70S BORONIR FLOWER VOLF1T ILES

METHYL .NAPHTHALENE (Peak 9) CV/ 1,4

•

03-DEC-80 22:13 CaL:1C300

CZ

at)

64

CS.■

METHYL N DECANOATE (Peak 10)

40

DEBR04 73e BORONIA FLOWER VOLATILES cfiL:1c3B0

Igo 200

DEBRO4 7S0 BORONI A FLOWER VOLATILES criL:Ic-3oo

UNKNoWN

03-DEC-80 Z3:12

16380

03-DEC-8011 2.2:38

■-■

I

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4' SS0

4 G9 -

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-

2 1 1

-I

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100

200

300

DEE3R131- 8S8 BORONIR FLOWER VOLHT ILES CAL:IC30

03-DEC-80 27:2 P.-IONONE (Peak 18)

zoo 30 0

FWOJ OFCLIRM

DIHYDROACTINIDIOLIDE (Peak 20) 1_

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IL

736 z

i.1 S7

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03—DEC-80 c8 27:SG3

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100

DEBRO4 883 BORONIA FLOWER VOLATILE cmL:lcam

- 181 -

1JE1036 92 PROSTRNTHERA LRSIFINTHOS °Vial 50-2404 CRL:10488 UNKNOWN

7

20-APR-82 2;54

180 ;■ .200 0E1036 117 PROSTRNTHERFI LFISIFINTHOS OV101 50-240 4

L:10480 STR: E. UNKNOWN 4

388 20-APR-82

3142

149

180 7-288— - 300 .

20-APR-82 3.S3

DE1036 123 PROSTFINTHERA LRSIFINTHOS OV101 50-240 4 L:10400 4 METHYL-PENT-1-EN-3-ONE

cc

27

942

380 20-APR-82

108 280

DE1036 1S3 PROSTFINTHERR LRSIANTHOS OV101 50-240 4 CRL: 10480 STR: E• UNKNOWN cc

4:S8

18S

40

- 182 -

0E1036 161 PROSTANTNERA LAS 1 FINTHOS OV101 50-240 4 20-APR -82 L:10480 STA:E. UNKNOWN s.s

4'4

1149

7

- - - , - 102 208

DE1036 166 PROSTANTHERA LAS FINTHOS OV101 50-240 4 CAL:10400 STR:E. UNKNOWN

4.4

• • 308

28-APR-82 5.14

) „J

574

4

2

a

1 00 208

0E1038 189 PROSTANTHERA LAS I ANTHOS OV101 50-240 4 L:12480 STA:E. UNKNOWN

20-APR-82 S :57

54

Id !IL J I

100 208 0E1036 243 PROSTANTHERF1 LASIFINTHOS OV101 50-240 4 CAL:10480 STA:E. PENTYL ESTER

ROB

20-APR-82 7.29

100 202 390

BENZALDHYDE •7

2 - 106

CAL:18460 STA:E.

2S8

4,1 107

542

121

,,. 1 , 11.1.1, , . 11. 100 200 300

- 183 -

0E1036 250 PROSTANTHERA LRSIRNTHOS OV101 50-240 4

109 DE1036 254 PROSTANTHERA LRSIRNTHOS OV101 sp-ate 4

L: 10400 STAE. c‹, -P 1 NENE

20—APR-82 7:S3

Jt_ J J - 1

1

-

108 288

300

I - C. C. C C

DE1036 267 PROSTRNTHERA LASIRNTHOS OV101 50 -240 4 L:10400 STA:E.

CAM PH ENE

22—APR-82 a.zs

4. 1" — 1 1

102 288 388

DE1036 296 PROSTANTHERR LASIFINTHOS OV101 50-240 4 22—APR-82

2163

41

G3

1

CAL sTA:E. :10480 9:29 -P I NENE "13

— 184 —

•20—APR-82 9144

meesaas PROSTANTHERA LASIANTHOS OV101 50-240 4

CRL:10480 STA:E. MYRCENE 4

189 /.289 0E1036 327 PROSTANTHERA LASIANTHOS OV101 S0-240 4

C1L:10408 STR:E. —PHELLANDRENE

- ---- - - - - 388

20—APR-82 10:18

•

664

20—APR -82 10126

DE1036 331 PROSTANTHERA LASIANTHOS OV101 50-240 4 CR1_:10408 STR:E. CYMENE MIXTURE

ct

4

111 1 189 - I :---- • -

228 - - -

CHElEr3G; 344 PROSTANTHERA LASIANTHOS OV101 50-240 4 CAL:1E3400 sTR:E. 1,8 CINEOLE + CYMENE

1 51

L ik iIJt

352

^-1 — - r

20—APR-82 101 S2

a

2132

134

k. . 100 209 390

4.1

-185 -

20-APR-82 11:11

0E1036 3SS PROSTANTHERF1 LASIRNTHOS OVi 01 50-240 4 CF1L: 113490 STR: E. UNKNOWN

4a

1.9

406S

■-•

, 203 360

7

34

IJI

DE1036 394 PROSTANTHERA LASIRNTHOS OV101 50-240 4 20-APR-82 L: 10490

SIR: E. UNKNOWN MOMOTERPENE

12 .24

136

216

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C C.

0E1036 404 PROSTRNTHERA LASIRNTHOS OV101 50-240 4 20-APR-82 CAL:IC/400 STR: E.

CIS LINALOOL EPDXIDE 12143

302

100 200 0E1036 420 PROSTRNTHERA LASIRNTHOS OV101 50-240 4

L:10490 STA:E. TRANS LINALOOL EPDXIDE Sc1

7

390 20-APR-82

13.14

a 4,1

132

s- -r 220

IJ

44

27 41

S'

84

t % 100

274

L 200

308

- 186 -

DE1036 437 PROSTANTHERA LASIRNTHOS OV101 S0-240 4 CRL:I0408 STA:E. L INALOOL 71

1100 0E1038 458 PROSTANTHERR LASIFtNTHOS OV101 Sle-;240 4 CRL: 10408

STR:E. UNKNOWN

- - ka1114- .1i .....

20-APR-82 13:46

784 •

-

398 20-APR-82

14125

100 •-••-•-•-•-•"1-•-• I - I

288 388

DE1036 460 PAOSTANTHERA LRSIRNTHOS OV101 S0-240 4 20-APR-82 CAL: 10400 STR:E. 14129

UNKNOWN Ft4

245

I , 180 288 288

0E1036 492 PROSTANTHER8 LASIANTHOS OV101 50-240 4 20-RPR-82 CAL: 10.408 - STR:E. CAMPHENE HYDRATE is,zs

4'4

4.1

3f.

20-APR-82 1S.43

0E1036 499 PROSTRNTHERA LRSIRNTHOS OV101 50-240 4 CAL:10488 sTR:E.

PINOCARVONE

' 0 ,

20-APR-82 16.11

1119

DE1036 514 PROSTRNTHERF1 LRSIFINTHOS OV101 50-240 4 CM:111400 STR: E • ARCHILLINOL (?)

s-4

388 20-APR-82

16.4S

1013 288 DE1036 532 PROSTRNTHERR LRSIRNTHOS 0V101 S0-240 4 CM:113480 SIR: E• TERPINENE-4-01

- 187 -

ea

2

e■—•

4

II r , . ,,_,u1 -

200 100

3SS2

33

86

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Z7

0,, ,,,,,„I. loik

,. 4 288 DE1036 SOS PROSTRNTHERR LRSIRNTHOS OV181 50-240 4 CRL:10488 STR.E.

27

208

UNKNOWN 4

•

288

28S

388 20-APR-82

is.s4

100 , 2013

DE1036 574 PROSTRNTHERA LRSIANTHOS OV101 50-240 4 CRI-:104021 STA:E. UNKNOWN

ctc

a

2 41 so

acao 20—APR-82

18:4

2-42

4854

43

93

I 121 3G

100 209

35

T Li

ISO

^ —

- 188 -

0E1036 548 PROSTANTHERA LRSIRNTHOS 0V101 50-240 4 20—APR -82 L:10400 STR:E. • TERPINEOL

17:IS

DE1036 577 PROSTRNTHERR LASIANTHOS OV101 50-240 4 1L: 10409 STR:E. UNKNOWN

20—APR-82 18110

215

STA:E. CARVONE

4

3r

"1-11 J111 - 0

DE1036 584 PROSTRNTHERR LAS1ANTHOS OV101 CAL:1[1400

-r 200 390

50-240 4 28—APR-82 18.23

971

8

20.

300 100

C - a

J

- 189 -

aa

0E1036 592 PROSTANTHERA LASIRNTHOS OV101 50-240 4 cAL:1o40e STA:E. UNKNOWN

54

it 1111, 100 -60

DE1036 603 PAOSTANTHERR LASIRNTHOS OV101 50-240 4 CFIL: 10400 sTR:E. UNKNOWN

20-APR-82 18:38

v. - -- e 308

20-APR-82 tsgss

6 -

121 a

00

27 150

till &at, 100

DE1036 GSS PROSTANTHERR LRSIRNTHOS 0 1/101 S0-240 4 cRL:104e0 STR:E.

I SO-BORNYL ACETATE 4-4

les DE1036 661 PROSTANTHERA LASIRNTHOS OV101 50-240 4

L:10400 • STA:E. UNKNOWN

./

200

I I I I T

ape 20-APR-82

20.4s

388

20-APR-82 28:37

322

2 - 43

3sa

109 2130 388

- 190 -

. . .

0E1036 670 PROSTANTHERR LASIANTHOS OV101 50-240 4 CRL:10480 STR:E. THYMOL

,

20-APR-82 21:5

13S

215

150

• I 1 - •- --- i• - I lea

13E1036 714 PROSTANTHERA LASIANTHOS OV101 S0=240 4 CAL:10400 UNKNOWN

4'4

TJ • -

380

20-APR-82 22=23

138

04 40

••••••" r•-• • ^' 'V • - - -

— — 390

-

• 0 0 • :

0E1036 719 PROSTANTHERF1 LASIANTHOS OV101 S0-240 4 20-APR-82

UNKNOWN CRL:10400 sTA: E 22:38

c7 181

1 I '

1 ''''•"•1 1 - ' -1 "

1 00 ---200 - 380

0E1036 76S PROSTANTHERA LASIANTHOS OV101 50-240 4 20-APR-82 L: 10498 • STA:E. UNKNOWN 245

7 496

132

Sr

HAL LA.

qpi 1,1 I g A _LAII 100 2B2

STR:E•

1 (

I,,,) 100 200

PROSTRNTHERA LASIANTHOS OV101 50-240 4 UNKNOWN

93 1137 133

121

100 -

208 380

20—APR-82 26:28

0E1036 841 PROSTANTHERA LASIANTHOS OV101 50-240 4 CAL: 10489 STR:E. UNKNOWN

100

GS

69

.acta

204

187 202

200

r -1 1t4L,

0E1036 823 CRL:10400

41

133

S37

161

204

280 320

0E1036 864 PROSTANTHERA LASIANTHOS OV101 50-240 4 20—APR-82 CAL: 1E1400 STA:E. UNKNOWN 26,s3

121

C — C

4.1

91

119 1 133 59

147

-

300 20—APR-82

WsS4

104

187

161 204

4

PRDSTANTHERA LASIANTHOS OV101 50 -240 4 STfl: E. UNKNOWN

o g -

0E1036 897 CRL:10400

308 20-APR-82

28s14

107 121

93

- 192 -

0E1036 857 PRDSTANTHERA LASIANTHOS OV101 50-240 4 L:113400 STR:E. UNKNOWN

20-APR-82 26.68

4) 1SS

27

L. 100 208

0E:1036 881 PADSTANTHERA LASIANTHDS OV101 50 -240 4 STR: E. CAL: 10488 UNKNOWN

•

I' r 300

20-APR-82 27&6

149

308

DE1036 Eaa PROSTANTHERA LASIANTHOS OV101 50-240 4 CM:111480 STR:E. UNKNOWN

20-APR-82 28.1

161

209

IL 699

7

121

41

107

4 7

SS

'300

214

308

161 189

?

134

- 193-

CAL: 11)408 .2ROSTANTHERA LASIRNTHOS OV101 50-240 4 sln:E. UNKNOWN c

20—APR-82 29:41

105

DE1036 960 CRL:1D488

PAOSTANTHEAA LASIANTHOS OV101 50-240 4 STR: E. UNKNOWN

• 390 20—APR-82

30.13

114

288

20—APR-82 30=24

0E1036 966 PROSTANTHEAA LASIANTHOS OV101 52-240 4 Cf1l.: 1E1480 STR: E • U NKNOWN

4'

111

172

LL.1,

0E1036 978 L: 10488

41

AJJ 11.11 1.41 J. 1 t tL___ , . 188 .288

PAOSTANTHERA LASIANTHOS OV101 50-240 4 STR:E. UNKNOWN

4-4

3 -

r 398

20—APR-82 3014?

2888

34

UL

101

208 388

644

11 1

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20-APR-82 -

DE1036 981 PROSTANTHERA LASIRNTHOS OV101 SO-240 4 CRL:10488 STR:E., UNKNOWN

4q.

41

159

, I 1,40, , ,L1)01,11it, JILL

, Lik

100 200 0E1036 38G PAOSTANTHERA LASIRNTHOS OV101 50-240 4 CAL: 10480 STR:E. UNKNOWN.,„.1

4:1

334

PA' Orb

161

„gm

G9

109 ----- — - - • -

200 390

a r •388

20-APR-82 31:2

•

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41

188

DE1 eras 1002 PROSTANTHERA CAL: 10480 STA:E.

2 41

LASIANTHOS OV101 50-240 4 20-F1PR-82 31:32

LEDOL 66S

1 ,300

100

149

- 195 -

DE1038 1007 PROSTANTHERA LF1S1ANTHOS OV101 50-240 4 20-APR-82 alt.: 10400 STFt: E. UNKNOWN

31142

4-4 203

41

107 a"

•I tot. ,

200 0E1036 1019 PROSTANTHERR LAS1ANTHOS OV101 50-240 4 CHU 11)408 sTR: E. -EUDESMOL

- ----- ----r 300

20-APR-82 32:4

100 200

— 196 —

DE1030 176 RADIATA BARK OIL CW2OM SO-200 DEG CRI.:1C3S0 STR:E. CAMPHENE

-0 19—JAN-81

. 61S

191

638

a - en

67

41

a

200 DE1030 226 RADIFiTA BARK OIL Cw20m S0-200 DEG CRL:1C3S0 sTR:E. PINANE ? .1c

320

19—JAN-81 7g48

408

6-7

138

- 100 200

en - g 300

1403

100

DE1030 231 RADIATA BARK OIL C12OM SO-200 PEG CAL:1t2S0 sTA:E.

3 CARENE

200 ' '300

390

1236

39—JAN-81 7:se

ct-1

DE1030 228 AROIATA BABK OIL CW20M SO-200 PEG CRL:1C3S0 STA:E.

UNKNOWN O ./

19—JAN—E l 7:SZ

- 197 -

0E1030 240 RADIATER BARK OIL CW20M SO-200 DEG cm:Ic3S0 MYCRENE

19—JAN—B1 817

100 200 300

0E1030 249 RADIATR BARK OIL CW20m S2-200 DEG 19—,M-81 CAL:1C3S0 STA:E. CYMENE (TYPE) s

ssts

91

—„Ia l 200 ' 300

-r

DE1030 2S7 RADIATA BARK OIL cw201 SO-200 DEG cm:Ic3s8 s1n: E. LIMONENE

13-JAN-B ]

8:52

1433

IL ,

0E1030 2S8 CFIL:IC3S6

L j • " " I • I

UNKNOWN

100

300 19—JAN—G]

8:54

100 zoo RADIATA BARK OIL cw20m SO-200 DEG STR:E.

2473

200 300

- 198 -

DE1030 271 RAD] ATA BARK OIL CW2OM SO-200 DEG cm:Icase STR:E.

g3 DODECANE

19-JAN-81 3.21

43 811

ro0

4

100 200

0E1030 273 RADIATA BARK OIL CW20M SO-200 DEG CRL:1C3S0 STS:E. PHELLANDRENE

300

19-JAN-81 9.2S

1920

,

-r 200

100

300

300

DE1030 278 RADIATA BARK OlL CW20M SO-200 DEG cm.:Ic3S0sTR:E. UNKNOWN

4'1

7

100 200

0E1030 313 RADIA1 A BARK OIL cw2er1 S0-200 DEG CRL:1C3S0 STR:E. M CYMENE

19-JAN--e) 9:3S

207

19-JAN-81 10:47

597

200 300

100

100 DE1030 371 ARDIRTA BARK OIL CW2OM S0-200 DEG

MYRTENOL

100

1 9-JAN-21 12:q6

353

137

152

200 300

CRL:1C350

- 199-

0E1030 321 RADIATA BARK OIL CW2OM S0-200 DEG CRL:1C350 STR:E.

io-CYMENE ils

19-JAN-81 11t 3

3970

101

134 911

100 2 I 1 1

00 300

DE1030 329 RADIATA BARK OIL CW2Om SO-20O DEG 19-JAN -81 CAL:1C3S0 STR:E. TERPINOLENE 11:z0

1?1

1039

136

200 300

100

1 9-JAN-2 2 12:1

DE1030 349 RADIATR BARK OIL CW2OM S2-200 DEG GRL:1C3S0 STR:E. C

13 HYDROCARBON + UNKNOWN

43 196

4

-

200 300

- 200 -

DE1030 396 RADIFITA BARK OIL CW2OM S3-200 DEG alL:Ic3se STR:E. UNKNOWN MIXTURE

19-JAN-8I 13,37

119

573

41

67

300 100 200

DE1030 397 RADIATA BARK OIL CW22M SO-200 DEG CRL:1C3S8 STR:E. UNKNOWN

19-JAN-81 13,40

719 `17

67

)61,,Atik tiN 11-

108 200

DE1030 403 RADIATA BARK OIL Cw20m S2-200 DEG CRL:1C3S0 STR:E.

I CYMENE (TYPE)

41

ig 300

I 9 - JRN-R 1 32:C2

327

13h

) ti )11 lilt 100 200

DE1030 416 RADIATA BARK OIL Cw20m S8-200 DEG : IC3S 0 SIB: E. UNKNOWN

100

300 '1

19-JAN-2, 14,13

137

953

kh r

200 300 T

188 200

DE12/30 438 RADIATA BARK OIL CW2OM SO-200 PEG cAL:Ic3S0 STA:E. FENCHONE

380 19—JAN-81

1514

69

41

1184

-[

100 200 388

- 201 -

923

DE10304E36 RADIRTA BARK OIL CW22M S2-200 DEG CRL:1C3S0 STR:E. -DIMETHYLSTYRENE

19—JRN-8 16e]

9

100 220 0E1030 481 RADIATA BARK Olt_ CW2OM SO-200 DEG CRL:1C3S0 STA:E.

474 UNKNOWN

19—JRN-81 16:32

132

4 67

10P Zeo 300

132

17

—

BORNYL ACETATE DE1030 430 RAD1ATA BARK OIL CW22M SO-200 DEG CRL:1C3S0 STA:E.

19—JAN—B] 14147

249

- 202 -

DE1 H30 484 RADIATA BARK DIL CW2Eiti S0-202 DEC CRL:1C3S0 STA:E. UNKNOWN

19—JAN-81 10:38

94

103 67

100

9.1

200 0E1030 435 RAM ATR BARK OIL C1420M SO-200 DEC CRL:1C3S0 STR:E. UNKNOWN SESQUITERPENE

9

htt -

300

19—JAN-81 • 17.1

167

10S 42

t iAiiLJ;

200 '300

14

19—JAN-83 17,1s

DE1030 S02 RADIATA BARK OIL 0,420m S2-2oe DEC CAL:1C3S0 STR:E. UNKNOWN

9 1194

41

-1/1'• I , 1100 200

DE1030 518 RAD I RTA BARK OIL Ct-120m S0-200 DEC CRL:1C3S0 STR:E. COPAENE

IS1

- f 300

1 9—JAN-8 I 17:46

176 IOS

:Ma

81 136

I I I I1 II I r ti 100

2/4

200 300 Jk I

1111IFerliptfl ,

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Set

9ZZ L ti

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00E 00Z 001

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tb • SS

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eoz eet CLI

1

DE1030 S88 RAD1ATA BARK OIL C1420m SO-200 DEC cRL:Ic350 STA:E. UNKNOWN SESQUITERPENE

79 41

1 8 200 300

19-JFIN-83 20:28

DE1030 S96 RFiDIATA BARK OIL Cw20m SO-200 _NKNOWN

DEG CFIL:1C3S0 STR:E. U

300

19-JAN-81 20:32

100 ZOO

DE1030 601 RRDIATA BARK OIL 042011 S3-200 DEC CRL:1C3S0 STR:E ISO BORNEOL

450

14- ‘3.38 200

- 204 -

19-JAN-8) 20.12

279

94

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A loll lb cAL:Icase STA:E.

109

133

300

19-JAN-81 • 202G

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UNKNOWN

011 8

c 7

S'A

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41

135 ige

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4S9

108

41

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1 9 —JAN-431 • 20.SS

19—JAN-8 2 20:S3

DE1030 GO8 RADIATA BARK OIL Cw20m 50-20O DEC CAL:1C3S0 STA:E. FENCHOL

41 89

107

STA:E. UNKNOWN CAL:1C3S8

19—JAN-81 21:s

0E1030 G14 RADIATA BARK OIL 0-120m S2-200 DEC CRL:1C3S0 SIR:E. 6 TERT. BUTYL CRESOL

- 205 -

200 300

4 SS)

164 —

.,-Ps cht. Rif , L 1 • I

I

100 610 DE1030 G22 AADIATA BARK OIL Cw20m S0-200 DEC caL:Ic3se STA:E. UNKNOWN

71

100

•1/41L A 188 208

DE103F3 G439 RADIATA BARK OIL CW2OM SO-200 OEG

200 300

4

300

19—JAN-81 21:22

100

1771

3303

CHL:1C3S0

leo . zoo DE1030 G62 RAD1 ATA BARK OIL Cw20ri S0-200 DEC

STF1:E. UNKNOWN

27

107

IS O 1 3 S

s r - 300

3 9—JAN-8 : 2244

64 3F.

1114

100

--r

200 3 r 00

200

- 206 -

DE1030 G28 RADIATA BARK OIL Cunti S0-200 DEC CRL:1C3S0 STA:E. TERPINENE-4-ol

71

I 9—JRN-8 21:34

923

4

,114..),LL

108 220

DE1030 G44 RADIFITA BARK OIL Cw20m S0-200 DEC

•• 300

1 9-3RN-8 1 GFIL:1C3S0 STR:E. 22s7 UNKNOWN 9

' 197 41

44

*

DE1 030 GS1 RRDIATA BARK OIL C42gm S0-200 DEG CAL:1C3S0 STA:E. MYRTENAL

IS—JANJ--Efl 2221

3

r100 200 r

300

DE1030 672 RADIATFI BARK Oil_ CL-120ti S2-200 DEG L:1635ø STR:E.

UNKNOWN 19-JAN-82

23.4

BORNEAL PLUS UNKNOWN ctc

CAL:1C3S2 STA:E.

—207 —

276

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134 Ga

g-

. 1141 rodtiviit. J J I ,

I

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302 100 200 DE1030 683 RADIATA BARK OIL CW20M SO-200 DEG 19-JAN-81

23.27

527

'

100 220 300

DE1030 631 RAOIATA BARK OIL CW2OM SO-200 DEG CAL:IC3S0 STA:E. UNKNOWN

19-JAN-81 23.43

•

317

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41. 67

217 Ji

'TB

1 Alit 11 1 L1I i,,t 4,1 I 300

19-JAN-81 24.0

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-36:24

A TECHNIQUE FOR THE SAMPLING OF VOLATILE ORGANIC COMPOUNDS

R. B. (Bob) Chesterman

R. (Bob) Chesterman, currently engaged in analytical and development work in the Department of Environment Laboratory in the Chemistry Department of the University of Tasmania, P.O. Box 252C, Hobart, 7001, Tasmania.

INTRODUCTION The sampling of volatile organic compounds has been undertaken using a wide variety of techniques such as cryogenic trapping', liquid adsorption 2, solid adsorption' and chemical reaction. Each technique has inherent problems associated with either the collection of unwanted material, such as moisture, or the quantitative desorption of components of interest. Tenax GC has proved one of the most effective trapping mediums and has been widely used for the collection of volatile organic compounds from 'air', waters and complex matrices including biological materia1. 6 . 2

The sampling system described has been used successfully to study volatile material from diverse sources including • plant material, industrial furnaces, working environment atmospheres, and for ambient air monitoring. Extensive development of systems outlined in the literature has resulted in a versatile sample collection/elution system for routine use in the field or industrial workplace. An outline of a similar system is given by Versinos in which he describes the use of 2.5 g Tenax sample tube to record recovery checks on a series of standard compounds. The capacity of Tenax to adsorb and desorb a range of organic components was reported in detail by Brown and Purne11 9 , and Butler and Burke.'° • Practical details of the sampling and desorption system used by this laboratory are given, and an example of the collection and analysis of volatiles from a wood burning stove is used to illustrate the technique.

EXPERIMENTAL PROCEDURES Sample collection tubes consist of 170 mm lengths of glass lined stainless steel tubing 6 mm 0.D., 4 mm ID., previously baked at 500°C and packed with Tenax-GC ( 0.4 g) and held by silanised glass wool. New packing was pre-conditioned at 325°C for 25 hours in a stream of high purity nitrogen, and the sample collection tubes were re-conditioned at 300°C for 2 hours prior to use in the field. The tubes were held individually in stoppered glass containers, with a small amount of dried silica gel, for storage and transport to and from the sampling area.

Collection of the volatile organic components of the flue gas was accomplished by inserting the Tenax sample tube into a water cooled jacket (Figure 1) and drawing gases through the sample tube from the flue of the wood burning stove. The sample collection point was 4 m above the grate. A calibrated flow control valve was used to maintain the gas flow rate to the gas pump at 40 mL per minute for 2 hours.

Flow

Pump control valve

Figure 1. Sample Collection System.

Desorption of the collected volatile organic components was controlled by heating of the sample tube in a stream of carrier gas to "inject" the trapped components into the inlet of a gas chromatograph or gas chromato-graph/mass spectrometer system (GC/MS) for characterisation.

The heating block consisted of a 25 x 150 mm cylindrical stainless steel rod with two holes drilled longitudinally. The central hole accepts the stainless steel Tenax sample tube held in position by a 1/4' Swagelock fitting at one end, and a 1/16' Swagelock fitting for the carrier gas inlet at the other. The second drilled hole contained a thermocouple junction to monitor the temperature of the block. The heating element consisted of a single layer of No. 20 gauge B. and S. resistance wire of

1.779 Ohm/metre. The total length of wire wound over a sheet mica base was 7.2 m. A voltage of 50 V from a variable voltage transformer provided sufficient power to heat the block to 300°C in approximately 10 minutes. Figure 2 is a half scale drawing of the elution equipment.

The eluted organic components are carried by the gas stream to a length of 2 mm x 230 mm glass lined stainless steel tubing (C) bent to form a U shaped trap. Coupling of the sample collection tube to the U trap was accomplished using stainless steel Swagelock fittings silver soldered to the 2 mm tube and employing graphite ferrules to form a compression seal at the heating block end. At each end of the 2 mm tubing (points A and B) a brass lug was silver soldered onto the fitting to provide electrical contact. Fitting A consists of a Luer syringe needle coupling or, an additional length of glass lined tubing, for coupling directly to a capillary GC col urn n.

Immersion of the U shaped section of tubing into a liquid nitrogen bath condensed the organics eluted from the heated sample trap. A 10 minute time interval at the pre-selected elution temperature ensured efficient displacement of the organics from the Tenax. After removal of the liquid nitrogen bath, the 2 mm glass lined tubing was rapidly heated by the Passage of an electric current (40A from a welding transformer) between points A and B. The current was applied for 30 seconds and the maximum temperature reached was 300°C. The organics were vaporised and carried to the injector system of the GC or GC/MS. Resistance heating of the glass lined tubing at this current setting is sufficient to ensure that breakage of the tubing does not occur.

Figure 2. Sample Elution — Injection Apparatus.

'tater -jacket and Tenax Samole Tube

Clean Air/August, 1982 39

2188.6

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0 100 200 6.50

300 4'00 13.36

t 500

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800

900 27.12

technique described could be accomplished on a routine basis using a number of sample tubes and despatching them to a central laboratory for analysis.

SUMMARY AND CONCLUSIONS An effective method for trapping volatile organic compounds from air and gas streams is presented. Construction details are provided and an example of the collection and an analysis of the flue volatiles from a wood burning stove is used to illustrate technique. Quantitative measurements of volatile components may be made by incorporation of a suitable bleed of a known concentration of a- specific component into the gas stream. Possible 0 applications of the technique include ambient air monitoring, forensic and customs search assistance, odour location and analysis.

Monitoring of volatile trace organic components in working and residential areas will become increasingly important as city populations increase. (There were 11 cities of over 1 million people in 1900, by the end of the century the number is expected to be over 500.) Trends in the lowering of ambient air quality and the health effects resulting need to be carefully documented at an early stage to avoid serious long term problems.

ACKNOWLEDGEMENTS The assistance of Mr. C. Richards in the construction of the equipment and the preliminary prototypes is gratefully acknowledged. The help of the staff and the use of the equipment of the Central Science Laboratory of the University of Tasmania is also acknowledged.

REFERENCES I. Giannovario, J. A., Grob, R. L., Rulon, P.

W., J. Chromatog. 121, 285, (1976). 2. Smith, R. G., Health Lab. Sc., 12, 167

(1975). 3. Rubinshtein, R. N. and Silchenko, S. A., ./.

Chromatog. 123, 251 (1976). 4. Pellizzari, E. D., Bunch, J. E., Berkley, R.

E., McRae, Anal. Chem., 48, 803 (1976). 5. Bellar, T. A., Lichtenberg, J. J., "The

Determination of Volatile Organic Compounds in Water by Gas Chromatography" EPA PubIn. EPA-670/4-74-009, Washington, DC (Nov. 1974).

6. Zlatkis, A., Lichenstein, H. A., Tishbec, A., Chromatographia, 6, 67 (1973).

7. Zlatkis, A., Bertsch, W., Lichenstein, H. A., Tishbec, A., Shunbo, F., Leibich, H. M., Coscia, A. M. Fleischer, N., Anal. Chem., 45, 763 (1913).

8. Versino, B., de Groot, M., Geiss, F., Chromatographia, 7, 302 (1974).

9. Brown, R. H., Purnell, C. J., J. Chromatog., 178, 79 (1979).

Table 1. Volatile Components from a Wood Burning Stove.

Component Boiling Scan

Point °C No.

I, 2-pentadiene 45 50 2, 3-pentadiene 48 55 acetone 56.5 57 hexane 70 63 benzene 80 74 2, 5-dimethylfuran 94 • 85 toluene 110 106 2-furyl methyl ketone 121 ethylbenzene 136 159 phenylacetylene 142 . 168 styrene 145-146 180 2-furaldehyde 162 218 benzaldehyde 179 236 dimethylphenol

(6 isomers) 210-225 247-248 p-methylbenzylalcohol 249 trimethylbenzene

(3 isomers) 164-170 253 benzofuran 173-175 287 naphthalene 217 573 azulene 270 574 dimethylbiphenyl

(5 isomers) 256-295 789 heptadecene 300 814 heptadecane 302 817

10. Butler, L. D., and Burke, M. F., J. Chromatog. Sc., 14, 117 (1976).

II. "Health Hazards of the Human Environment", W.H.O. Publn., Geneva, p.24 (1972).

12. "Hazards in the Chemical Laboratory", Muir, G. D., Ed., Chem. Soc. Publn., London (1977).

Table 2. Threshold Limit Values (TLV) for some Volatile Components

Component TLV (ppm) (mg/m 3 )

hexane 100 360 benzene 10 32 acetone 1000 2400 toluene 100 375 ethylbenzene 100 435 xylenes 100 435 furfural 5 20 - styrene 100 420 cresols 5 22

Table 3. Volatile Components of Boronia Headspace

Scan No. Identity

41

Carbon dioxide 80

Methylene chloride 01

Chloroform 10

Methylcyclopentane 23

Cyclohexane 40

Heptane 51

Methylcyclohexane 70

Toluene 200

Tetrachloroethylene 241

Xylene 293

Nonane 342

Monoterpene (unknown) 349

Isopropylbenzene 371 a-pmene 386

Camphene 392 ,3-pinene 400

Myrcene 416

Limonene 442

Linalool 522

Unknown 574

Menthone 600

Naphthalene 604

Ethylbenzene 705

Methylnaphthalene 736

Methyldecanoate 858

0-ionone 882

Dihydroactinidiolide

Figure 4. Total Ion Current Trace Boronia Volatiles Scan Number and Time.

100 -

1000 1100 Scan numbers

30.13 Time (minutes) .-

Clean Air/August, 1982 41

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