masters' diploma in technology (chemistry)i the development of a method for the analysis of...

\I

THE DEVELOPMENT OF A METHOD

FOR THE

ANALYSIS OF MAHEWU

BY

Richard G de Goede

submitted in fulfilment otthe requirements for the

MASTERS' DIPLOMA IN TECHNOLOGY

(CHEMISTRy)

in the

DEPARTMENT OF' CHEMICAL SCIENCES

of.. I

TECHNIKON NATAL

Supervisors: Dr J.H. Adamson (Technikon Natal)

Dr D.N. Neethling (Technikon Natal)

TITLE: The Development of a Method for the Analysis of

Mahewu.

AUTHOR: Richard Goodwin de Goede

Dissertation submitted in compliance with the requirements

for the Masters' Diploma in Technology (Chemistry) in the

Department of Chemical sciences at Technikon Natal,

Durban.

DATE OF SUBMISSION: January, 1994

I hereby declare that this dissertation represents my own

work both in conception and execution.

R.G. de Goede, DURBAN, 1994-01-30

Approved for final submission

Dr J.H. Adamson, Supervisor

ii

TABLE OF CONTENTS

ABSTRACT 0 • 0 • 0 • 0 ••• 0 • 0 0 0 0 ••••••• 0 • 0 ••• 0 •• 0 iii

SUMM.ARY • 0 0 0 0 0 0 0 0 •• 0 0 0 • 0 0 0 0 0 0 0 0 0 0 0 0 0 0 • • • • • iv

SUMMARY IN AFRIKAANS..................... vi

ACKNOWLEDGEMENTS......................... viii

ABBREVIATIONS • • • .0. • • • • • • • 0 0 • • • • • 0 • • • • • •

CHAPTER 1 INTRODUCTION • 0 0 0 • • • • 0 0 • 0 0 1

CHAPTER 2 HEADSPACE ANALYSIS • • 0 0 000 4

CHAPTER 3 SOLVENT EXTRACTION o • 0 0 0 0 0 14• CHAPTER 4 ANALYSIS 26• • • • 0 0 • • • • 0 0 • 0 • 0 0

CHAPTER 5 DISCUSSION o • • 0 • 0 • • • • • • • 0 0 43

ix

LIST OF REFERENCES ............•......... 49

APPENDIX o. 0 0 ••••• 0 0 0 0 •• 0 0 0 0 • 0 ••• 0 0 •• 0 • •• 51

iii

ABSTRACT

A comparison was made of various methods for the analysis

of the odoriferous components of Mahewu, a fermented

mealie meal porridge. The most satisfactory procedure was

found to be that of dynamic headspace sampling. This

technique, used in conjunction with gas chromatography and

mass spectrometry, allowed the positive identification of

several components .

•

iv

SUMMARY

Mahewu (also known as amahewu and magou) is a non-alcoholic

beverage drunk by African people. It is made by fermenting a

runny mealie meal gruel (porridge). The resulting .ferment is sour

to the taste because a product of the fermentation is lactic

acid.

•A comparison was made of various methods for the analysis of the

odoriferous components of Mahewu. The most satisfactory

procedure was found to be that of dynamic headspace sampling.

This technique, used in conjunction with gas chromatography and

mass spectrometry, allowed the positive identification of several

components.

The dynamic headspace sampling was performed to concentrate the

odoriferous components on a Tenax column, using a Tekmar

Headspace concentrator for small samples. Samples were also

taken from the manufacturer's tanks, but the water vapour in this

headspace made this procedure unsatisfactory. This sample was

then transferred as a plug to a GC column where it was separated

in the GC. The dynamic headspace concentration process had to

be optimised in order to produce a satisfactory response in the

detector. The GC conditions had to be optimised to produce

satisfactory separation of the components, hence resolved peaks

on the chromatograms. A mass spectrometer was used as the

detector. The mass spectra data were stored on a computer data

disc and the spectral data for each peak compared with the mass

spectra of compounds in the library in the computer. This

library search yield three possibilities for each reasonable

peak. These were studied, together with properties of the

vi

OPSOMMING

Mahewu (ook bekend as amahewu en magou) is 'n nie-alkoholiese

drank wat deur swartmense gedrink word. Dit word gemaak deur In

loperige mieliemeelpap te laat fermenteer. Die gevolglike ferment

smaak suur want een van die produkte van fermentasie is melksuur.

In Vergelyking van verskillende metodes vir die ontleding van die

reukgewende komponente van Mahewu is gemaak. Monsterneming van

die dinamiese kopruimte is as die mees bevredigende prosedure

bevind. Tesame met gaschromatografie (GC) en massaspektrometrie

(MS) maak hierdie tegniek die positiewe identifisering van

verskeie komponente moontlik.

Monsters van die dinamiese kopruimte is gene em om die reukgewende

komponente met behulp van 'n Tekmar-kopruimtekonsentrator vir

klein monsters op 'n Tenax-kolom te konsentreer. Monsters is ook

in die vervaardiger se tenks geneem, maar as gevolg van die

waterdamp in die kopruimte was hierdie prosedure onbevredigend.

Die monster van die Tenax-kolom is toe in gekonsentreerde vorm

na 'n GC-kolom oorgedra waar dit in die GC afgeskei is. Die

konsentrasieproses van die dinamiese kopruimte moes

geoptimaliseer word om 'n bevredigende reaksie in die detektor

te verkry. Die gaschromatografiese toestande moes ook

geoptimaliseer word om 'n bevredigende skeiding van die

komponente te verkry, vandaar die geskeide pieke op die

chromatogramme. 'n Massaspektrometer is as die detektor gebruik.

Die massaspektradata is op 'n rekenaardataskyf vasgele en die

spektrale data vir elke piek is met die massaspektra van

verbindings in die rekenaar se biblioteek vergelyk. Hierdie

proses het drie moontlikhede vir elke redelike piek opgelewer.

v

sUbstances. In this way the number of possible substances was

decreased. Because of their availability, commercial samples of

six of these were obtained and used to positively identify three

of the components. The positive identification could not be

performed using the MS because it ceased to function so a flame

ionisation detector was used instead, the components being

positively identified by spiking a mahewu sample with the

standards in turn. The components that were positively identified

were ethyl acetate, ethyl propionate and propyl benzene.

An attempt was made to quantify these components in a mahewu

sample but dynamic headspace sampling does not lend itself to

consistent quantitation.

•

Several solvent extraction techniques were investigated to check

whether the odoriferous components in the headspace could be

isolated from the gruel and thus concentrated in the solvent.

The solvents tried, which ranged in polarity, included

dichloromethane, methyl ethyl ketone, hexane, ether and sunflower

oil that is used for cooking. Portions of these extracts were

admitted to the GC/MS for separation and identification, but none

of the solvents nor methods proved satisfactory.

vii

Die moontlike stowwe, tesame met hul eienskappe, is bestudeer.

Die getal moontlike stowwe is sodoende verminder. Kommersiele

monsters van ses van hierdie stowwe is vanwee hul bekombaarheid

gekies en gebruik om drie van die komponente positief te

identifiseer. Die massaspektograaf het onklaar geraak en kon dus

nie gebruik word om die positiewe identifikasie te doen nie. 'n

Vlamionisasiedetektor is in plaas daarvan gebruik en die

komponente is positief geYdentifiseer deur die standaarde om die

beurt by 'n mahewu-monster te voeg. Die positief geYdentifiseerde

komponente was etielasetaat, etielpropionaat en propielbensien .

• Daar is gepoog om hierdie komponente in 'n mahewu-monster te

kwantif iseer, maar monsterneming van dinamiese kopruimte leen hom

nie tot konsekwente kwantifisering nie.

Verskeie extraksietegnieke met oplosmiddels is ondersoek om vas

te stel .of the reukgewende komponente in die kopruimte van die

dun pap afgeskei kon word en sodoende in die oplosmiddel

gekonsentreer kon word. Oplossers van wisselende polariteit is

probeer, insluitende dichlorometaan, metieletielketoon, heksaan,

eter en sonneblomolie (vir kookdoeleindes). Gedeeltes van hierdie

ekstrakte is in die GC/MS vir afskeiding en identifisering

ingevoer, maar geeneen van die oplossers of metodes het

bevredigend geblyk te wees nie.

viii

ACKNOWLEDGEMENTS

I would like to express my appreciation to Dr J.H. Adamson for

his invaluable assistance and support while he acted as my

supervisor as well as to mY,co-supervisor, Dr D.N. Neethling,

especially for her encouragement.

I would also like to thank Dr H. Greenberg for starting me off

on the project and for supervising in the early stages.

• I wish to thank Dr A.A. Spark for all his help, especially on the

GC/MS.

I thank those members of staff of the Department of Chemical

Sciences who helped in many ways.

I would like to thank the staff of Clover Dairies who supplied

samples and allowed me to sample their tanks.

•I am grateful to Mr B Parel of the Department of Chemistry at the

University of Natal for his help in printing many of the

chromatograms.

Finally, I acknowledge the financial support of the FRD and of

Technikon Natal, whose support enabled this work to be performed.

ix

ABBREVIATIONS

GC

GC/MS

Tekmar

FlO

Gas Chromatograph(y}

Gas Chromatograph(y)/Mass Spectrometer(y)

Tekmar Headspace Concentrator

Flame ionization detector

degrees Celsius

millimetres

centimetres

centimetres cubed

decimetres cubed

alcohol (ethanol)

diethylether

2-propanone (acetone)

benzene

trichloromethane (chloroform)

dichloromethane (or methylene chloride)

2-butanone (or methyl ethyl ketone)

diethyl ether

revolutions per minute

microlitre

rom

cm

em'

dm3

al

eth

ac

bz

chI

OCM

MEK

ether

rpm

#-£1

1

CHAPTER 1

INTRODUCTION

•

101 MAHEWU

Mahewu (also known as amahewu and magou) is a non-alcoholic

beverage drunk by African people. It is prepared by firstly

making a fairly runny mealie meal porridge, then adding some

bacteria-containing sUbstance which ferments the mixture

producing lactic acid. The lactic acid imparts a sour taste to

the porridge. The traditional lactobacillus-containing additive

is wheat flour. Mahewu is also produced on a large scale in

industry, the lactobacillus having been developed in order to

reduce the souring time from about 36 hours using the traditional

method to a matter of 3 hours. This also has the effect of

ensuring that quality is more uniform from batch to batch as well

as eliminating undesirable products such as ethanoic acid and

butanoic acid. 1

102 REASON FOR THE INVESTIGATION

The project originated when one of the manufacturers of mahewu,

Clover Dairies, encountered a problem on their plant in Congella.

Every now and again they would make a batch which the human

tester at the end of the line would reject. Testing was entirely

subjective using the nose. Seeing that each batch was

2000 litres, such rejection was costing the manufacturer a large

2

amount of money. It was not known what caused the off-odour

which led to rejection of the batch. It was decided to attempt

to isolate and identify the offending gaseous sUbstance so that

the microbiologist could attempt to decide what was producing

this substance. A strategy for dealing with the contamination

could then be developed. However, before the basic profiles of

uncontaminated mahewu could be ascertained, the manufacturer

moved to a new site where they had installed a new mahewu plant

in Queensburgh. The off-odour was no longer a problem so the

need to identify the offending substance disappeared.

The main thrust of the project changed to analyzing the headspace

of mahewu, without trying to identify off-odour components. If

basic profiles of the headspace could be established under given

analysis conditions then, if an off odour occurred in a batch,

this would facilitate the identification of the peaks due to

these undesired components.

103 SUMMARY OF WORK ALREADY DONE IN THE FIELD OF ANALYSIS OF THE

OLFACTORY COMPONENTS OF MAHEWU

In the field of the analysis of the olfactory components of

mahewu there is no work documented in the literature covered by

Chemical Abstracts. However, the manufacturers that had the

problem did supply a report of a headspace analysis done by PA

Torline of the National Food Research Institute, CSIR2. Details

of the procedure were not supplied, only a list of possible

3

components as determined by GC/MS analysis.

Because lactic acid, CH3 - CH(OH) - COOH, is a product of the

fermentation it was expected that propanoate esters were likely

to be present in mahewu. The lower molecular mass esters are

sweet-smelling and volatile ..

In dealing with the olfactory components one of the main problems

is that some substances produce a very strong response in the

olfactory receptors even in extremely low concentrations, e.g.

hydrogen sulphide, whereas other substances produce an extremely

small or no response even in fairly high concentrations.

Therefore in the headspace of mahewu there are likely to be

components in large concentrations that do not contribute to its

smell. Also there are likely to be components in extremely small

concentrations that contribute greatly to its smell3•

A great deal of work has been performed on the analysis of

headspaces above many similar beverages. "Several methods have

been used for concentration. Cryogenic techniques (e.g. cold

traps, freezing of sample in empty capillaries, direct

introduction in cooled open tubular columns), adsorption on solid

adsorbents (charcoal, silicagel, alumina, porous polymers) and

conventionally coated GC supports have been applied.,,4

Because of the availability of a headspace concentrator and a

GC/MS it was decided to follow this path in the analysis of the

headspace.

4

CHAPTER 2

HJEADSPACE ANALYSIS

201 INTRODUCTION

Some preliminary investigations were required to establish flow

rates in the sampler and in the GC capillary column.

Because of the low concentrations of the odoriferous components

of the headspace a technique for concentrating them was

necessary. Dynamic headspace sampling is the continuous removal

of the headspace vapour above a liquid by means of an inert gas

flow with subsequent trapping of the sample components by

adsorption or cold trappings. Trapping was done by adsorption

by a Tenax column. The GC/MS was connected to the dynamic

headspace sampler in a way such that the carrier gas supply line

went through the sampling instrument, but bypassing the sample

and adsorbent, then lead through a heated line to the injection

port of the GC. When the sample on the adsorbent is desorbed a

valve enables the carrier gas to be diverted over the flash-

heated adsorbent, thence to the GC.

5

202 EQUIPMENT

Gas Chromatograph: Varian 6000

Headspace Concentrator: Tekmar Model 4000 Dynamic Headspace

Concentrator

Tekmar Model 4100 Heated Sampler Module

GC/MS: Finnigan 1020

When the Tekmar is connected to the Finnigan GC/MS the set of

equipment is known by the Environmental Protection Agency as the

'Organics in Water Analyzer'.

203 INSTRUMENT CONDITIONS

Tekmar Headspace Concentrator:

Purge time 10 min. (unless otherwise

specified)

Purge ready temperature

Desorbtion temperature

Purge flow rate

30°C

150°C

40 cm3/min.(unless otherwise

specified)

Desorbtion time

Baking temperature

Baking time

0.2 min.

225°C

10 min. (unless otherwise

specified)

6

GC:

Carrier gas, nitrogen 4 cm3/min.

Make-up gas, 'nitrogen 30 cm3/min.

Detector FlO

Air flow rate 300 cm3/min.

Hydrogen flow rate 30 em3/min.

Injector temperature 120 °c

Detector temperature 250 °c

GC/MS:

Zone temperature 280°C

separator oven temperature 240 °c

Manifold temperature

Current injection mode

Splitless time

Turn filament off at

Current filament mode

Scanning

Scan rate

Threshold

Min. peak area

80°C

Capillary

o

o

A

30 - 600

0.95 s

2

20

Temperature program (unless otherwise specified):

Initial temperature

Final temperature

Initial time

Ramp rate

Final time

40°C

170°C

4 min.

5 °C/min.

10 min.

7

GC columns

The following commercial open tubular capillary columns were

used:

BP-1: 25 m. A non-polar column containing 100% methyl silicone.

BP-5: 30 m. A non-polar column containing 5% phenyl methyl

silicone.

BP-20: 25 m. A polar column containing Carbowax 20M.

PEG-20M: 25 m. A polar column (equivalent to the BP-20)

containing polyethylene glycol 20M~•2o~ PRELIMINARY INVESTIGATIONS

The optimum gas flow rate in the capillary column was ascertained

by performing a Van Deemter plot.6 From this a carrier gas flow

rate of 4 cm3jmin was found to be satisfactory, realising that

with capillary columns the Van Deemter plot is much flatter than

for a packed column. Therefore it is not necessary to use a flow

rate that is very close to the optimum as determined by the

Van Deemter plot.

205 PRELIMINARY RUNS FOR DETERMINATION OF CONDITIONS

In these runs the Tekmar was connected to the GC. The column used

was the BP-1. At this stage the sample heater was in operation.

The sparge tube was positioned 5 mm above the liquid level of the.

sample. It was not bubbled through the sample because when it

was bubbled through too much foam was produced. A 5 min preheat

8

and purge flow rate of 40 cm3/min. with an attenuation setting of

32 x 10-12 were used on the following and the corresponding

chromatograms obtained as shown:

Blank sample tube at 80°C, 5 min. purge (CHROMATOGRAM 1)

The same tube repurged at 80°C, 5 min. purge

(CHROMATOGRAM 2)

5 cm3 mahewu at 80°C, 5 min. purge (CHROMATOGRAM 3)

5 cm3 mahewu at 80°C, 10 min. purge (CHROMATOGRAM 4)

5 cm3 mahewu + 3 g sodium chloride at 80°C, 10 min. purge

(CHROMATOGRAM 5)

Because of the differences in the chromatograms obtained for the

purges of the blank tubes it was decided that all sample tubes

would be heated in an oven at 120°C for at least one hour,

before being used.

The sodium chloride was added to saturate the mahewu sample to

see if this would have a marked effect on the decrease of the

solubilities of the headspace components, thus increasing the

concentration of the components in the headspace. The peaks were

only a little larger so it was decided that sodium chloride would

not be added to future samples.

9

206 EFFECT OF TEMPERATURE OF MAHEWU SAMPLE ON THE HEADSPACEo

Using the temperature program:

Initial temperature

Final temperature

Initial time

Ramp rate

50°C

250°C

10 min.

10°C/min.

•Final time 2 min.

chromatograms were obtained for the sample at room temperature

and at 90 °C (CHROMATOGRAMS 6 & 7).

The chromatograms show marked differences in the number of peaks

and in the heights of common peaks. Because the mahewu is

usually consumed at ambient temperature all further headspace

work that was carried out on it in the laboratory was performed

at room temperature.

207 EFFECT OF PURGE TIME ON CHROMATOGRAMS

Considering CHROMATOGRAMS 3 & 4 it was decided to purge all

further mahewu samples at 40 cm3/min. for 10 minutes when using

the Tekmar, employing a similar temperature programme to ensure

that all components are eluted before the next injection.

ALL SUBSEQUENT QUALITATIVE ANALYSES OF THE HEADSPACE WERE

PERFORMED USING THE TEKMAR CONNECTED TO THE GC/MS.

10

From time to time the source of the MS was cleaned, taking all

the necessary precautions as prescribed in the manufacturers'

manual.

A number of runs were done on various mahewu samples (Files

DEGOEDE02 through to DEGOEDE37). The analysis of the headspace

components of these is used in a later chapter.

208 LONG PURGES

It was decided that it would be desirable to sample the headspace

of the producer's tanks. This necessitated the construction of

a portable sampling apparatus. Tenax was conditioned by placing

it in the oven at 120°C for 3 hours. The ends of 90 mm glass

tubes with inside diameters of 5 mm were fire glazed and each

packed with 0.15 g of Tenax. The Tenax was held in each tube by

plugs of glass wool.

For each long purge performed in the laboratory nitrogen from a

cylinder was passed through 600 cm3 of mahewu sample in a 1 dm3

glass bottle fitted with stainless steel inlet and outlet tubes,

thence through the tube containing Tenax. The nitrogen thus

bubbled through the sample for 48 hours. The Tenax with the

adsorbed components was transferred to a clean sample tube and

fitted to the Tekmar. Because the heater unit was not working

the sample tube containing the Tenax was heated using a spirit

burner while being purged with nitrogen at a flow rate of

12 cm3/min. for 4 min. Desorption was for 0.5 min. at 135°C.

12

209 TANK PURGING

Having developed a method for long purges in the laboratory a

starter tank at the suppliers' plant was sampled-by drawing the

headspace directly from the tank using a portable pump which

pumped at a set rate of 1 dm~/min. A special stainless steel

plate had to be made to fit the sampling port on the tank and to

facilitate the tubing to the Tenax tube thence to the pump. The

headspace above the tank was thus sampled for 1 hour. Back in

the laboratory the Tenax was removed with difficulty from the

tube. The process was difficult because the Tenax was wet from

the water vapour in the headspace above the hot mahewu in the

tank. The same conditions were used to produce

CHROMATOGRAM 12. The chromatogram showed evidence of column-

bleed caused by water vapour.

A purge of the starter tank was repeated with a drying tube in

line before the Tenax tube. The drying tube consisted of a U-

tube, of inside diameter 2 cm, containing anhydrous sodium

sulphate with glass wool keeping the drying agent in place. This

did not prove satisfactory because the sodium sulphate and glass

wool removed from the tube had a definite smell, indicating that

odoriferous components were not reaching the Tenax in their

entirety.

11

The line heater controller was not operational so this was done

manually.

The temperature program used for this run was:

initial temperature 60°C for 4 min.

ramp rate 5°C/min.

final temperature 200°C for 10 min.

and CHROMATOGRAM 8 obtained.

• To check if any artifacts remained on the original Tenax, the

sample tube was heated in an oil bath at 150°C during the purge

cycle. For this run a final temperature of 230 °C was employed

and CHROMATOGRAM 9 obtained.

The same mahewu was purged again after 37 days, this time purging

with nitrogen at 12 cm3/min. for 36 hours. The adsorbed material

was delivered to the GC/MS as before except that the sample tube

on the Tekmar was heated in an oil bath at 150°C, resulting in

CHROMATOGRAM 10.

with a fresh mahewu sample and the same procedure as for

CHROMATOGRAM 10, CHROMATOGRAM 11 was obtained.

One problem associated with the GC/MS was that if the mains

voltage dipped during a run the computer automatically reset the

instrument. This occurred during the chromatographic run of a

36 hour purge of a home-fermented sample of mahewu, resulting in

the loss of all the data.

13

At this stage no more work w~s possible on the GC/MS because it

has not worked satisfactorily since then. The electrometer was

faulty, then when it was fixed it was not possible to tune the

MS satisfactorily. At one stage the disc drive damaged someone

else's data disc, necessitating a shutdown of some time until

repaired. The printer required for the output of chromatograms

ceased to function so this had to be done on an identical system

at another institution.

14

CHAPTER 3

SOLVENT EXTRACTION

301 XNTlRODUCTION

Because the headspace components originate from the mother liquor

it was decided to attempt to extract these compounds from mahewu

using solvent extraction techniques, to separate them and

identify them. In this way it may be possible to identify off-

odour compounds without working on the headspace.7

The solvents were chosen because of their range of polarities.

Their properties are shown in the following table.8

TABLE 301

SOLVENT OEN.SITY at BOILING POINT

200c/g em? 1°C

OCM 1,325 40

MEK 0,806 79

Hexane 0,659 69

Ether 0,714 35

The concentration of the extracts in the solvents may be

15

increased by rotary evaporation or by passing a stream of inert

gas over the surface to encourage evaporation of the solvent.

The inherent dangers involve attrition of very volatile solutes

and possibly including impurities from the gas stream.9

After seeing a paper by Dupuy et alto on the use of vegetable oil

to extract flavour components this basic idea was used on mahewu.

302 SOLVENT EXTRACTION METHODS

30201 with solvents less dense than mahewuo

The apparatus shown in FIGURE 1 was used.

16

FIGURE 1

condenser

[ 100 mID

o nunsolvent

Here the solvent in the round-bottomed flask was heated with a

heating marrtLe, the vapour condensed in the condenser collected

in the funnel, dropped down the tube and rose through the

sintered glass disc thence through the mahewu sample. Excess

solvent that contained extracted components dripped back into the

heated vessel, this all in a continuous cycle.

17

50 cm3 mahewu was extracted for 3 hours with 300 cm3 hexane. A

60 cm3 aliquot of the hexane portion was concentrated to 10 cm3

on a rotary evaporator at 100 rpm, i.e. a concentration of 6

times. 0.5 ~l of this concentrate was injected into the GC and

CHROMATOGRAM 13 obtained using split injection and the SE-52

capillary column.

0.5 ~l of the neat hexane, concentrated in the same way, was

injected and CHROMATOGRAM 14 obtained. This was done to ensure

that any peaks in the chromatogram were not confused with peaks

due to impurities in the hexane. It was seen that the hexane

contained very few impurities. This was the case with all

solvents used.

The same extraction was repeated for 8 hours and the resulting

CHROMATOGRAM 15 obtained.

These chromatograms showed that the longer the extraction the

greater the concentration of the extracted components in the

extracting solvent as shown by the greater height of the peaks.

It was therefore decided to perform 8 hour extractions.

An 8 hour extraction using hexane, concentrated 20 times produced

CHROMATOGRAM 16. Comparing CHROMATOGRAMS 15 & 16 shows that the

greater the concentration the greater the size of the peaks and

that the rotary evaporation method of concentration is

satisfactory.

18

Similar extractions were performed with MEK, using the same

procedure. With the PEG-20M column, the following chromatograms

were obtained:

CHROMATOGRAM 17

CHROMATOGRAM 18

hexane

hexane, 8 hour extract,

concentrated 20 times

MEK

MEK, 8 hour extract, concentrated

20 times

CHROMATOGRAM 19

CHROMATOGRAM 20

In the run for chromatogram 18, after 4 min. the attenuation was

changed to a more sensitive value to see if there were any other

components in small quantities eluting after that time; some

peaks are seen.

Using the GC/MS and the BP-1 column, the following chromatograms

were obtained with 2 ~l injections:

CHROMATOGRAM 21 hex a n e, 8 h 0 u rex t r act ,


(Filed as ROG1)

CHROMATOGRAM 23


unconcentrated (Filed as ROGEl)



(Filed as ROGE2)

MEK, 8 hour extract, concentrated

20 times (Filed as ROG2)

CHROMATOGRAM 22

CHROMATOGRAM 24

19

An ether extract was performed by using 400 cm3 mahewu with

40 cm3 ether and 250 cm3 water in the boiling flask. The

continuous extraction was performed for 8 hours.

An ether blank was obtained by evaporating to dryness a 15 cm3

aliquot of ether by blowing a stream of nitrogen over the surface

of the liquid. 100 ~l of ether was added and swirled around the

inside of the vessel, i.e. concentrating the extract 150 times.

0.5 ~l of this was injected., producing CHROMATOGRAM 25.

The same concentration procedure was used for the ether extract,

producing CHROMATOGRAM 26. Because the peaks after the initial

large solvent peak were so small another injection was made using

the splitless mode, resulting in CHROMATOGRAM 27, which shows

that this is a much better technique, but still very

disappointing because they are not well resolved, still

relatively small and very close to the very large solvent peak

at the beginning of the chromatogram.

• This concentration procedure was used for a new hexane extract

in which 400 cm3 mahewu and 200 cm3 water was extracted with

50 cm3 hexane for 8 hours. A blank was obtained,

CHROMATOGRAM 28 (File HEXANE01) by evaporating 15 cm3 of the

hexane to dryness and adding 100 ~l hexane. 4 ~l was injected

using splitless mode for 30 s and the following temperature

programme:

20

Initial temperature 60 °C

Final temperature 230 °C

Initial time 5 min.

Ramp rate 5 °C/min.

Final time 5 min.

The same concentration procedure was used for the hexane extract

to obtain CHROMATOGRAM 29 (File HEXANE03). The evaporated

•extract had a strong odour so the odoriferous components must

have come out with the solvent peak. In order to confirm this,

another aliquot of the hexane extract was evaporated and an

injection made with the residue to obtain CHROMATOGRAM 30

(File HEXANE07). The two peaks are due to odoriferous

components. Their quantities' are extremely small as shown by the

ion count.

30202 with solvents more dense than Hahewuo

The apparatus shown in FIGURE 2 was used.

21

FIGURE 2

condenser

i

100 nun

•

o nun

The sol vent was boiled using a heating mantle. The vapours went

up to the condenser, condensed and dropped through the mahewu.

Excess solvent that contained extracted material overflowed back

into the boiling flask, the process thus cycling continuously

during the course of extraction.

22

In this way, 30 cm3 mahewu was extracted for 4 hours using

200 cm3dichloromethane. 125 cm3 of the dichloromethane extract

was concentrated on a rotary evaporator to 5 cm3, i.e. 25 times.

0.5 J.Ll dichloromethane was injected and CHROMATOGRAM 31 obtained.

Likewise, CHROMATOGRAM 32 was obtained for the extract.

A similar extraction was carried out for 8 hours, the extract

concentrated 30 times, yielding CHROMATOGRAM 33. with this

method of concentration it was difficult to stop the evaporation

at the same point each time . However, this could have been• improved by adding a little solvent to make it up to the same

volume each time.

30203 oil extracts

The vegetable oil used for these extracts was Helios Cooking oil,

a sunflowerseed oil.

The sample heating unit on the Tekmar was operational for these

analyses.

A blank was obtained, CHROMATOGRAM 34 (File 1), by placing

0.2 cm3 oil on glass wool in a clean sample tube, connected it to

the Tekmar and purged at 100°C for 10 min. before desorbing on to

the GC/MS using split mode and the following heating programme:

Initial temperature 40 °c


Initial time 4 min.


Final time 10 min.

23

The oil extraction was effected by stirring together 10 cm3 oil

and 100 cm3 mahewu on a magnetic stirrer for 1 hour. The

resulting mixture was centrifuged and separated. 0.2 cm3 of the

oil extract was placed on glass wool in a clean sample tube,

heated to 100°C and purged. The trapped components were

forwarded to the GC/MS to produce CHROMATOGRAM 35 (File 2).

•Another 0.2 cm3 of the oil extract was placed on glass wool in a

clean sample tube, heated to 40°C, purged and chromatographed,

giving CHROMATOGRAM 36 (File 3). The oil extract was left on the

glass wool in the sample tube and the same procedure repeated at

50°C, 60 °c and 70°C to see if any other peaks appeared. The

following were obtained:

50°C:

60 °C:

70°C:

CHROMATOGRAM 37, File 4



Comparing the above chromatograms it seems that 60 °c would be a

suitable temperature to use and that only the component giving

the peak at about scan 300 could be studied.

Another extraction was performed using 20 cm3 oil and 200 cm3

mahewu.

An oil blank was run using 1 cm3 of the oil on glass wool at

40°C (CHROMATOGRAM 40, File 11).

1 cm3 oil extract on glass wool was run at 40°C

(CHROMATOGRAM 41, File 12).

Comparing Chromatograms 36 & 38 it is seen that the peaks of the

latter are larger. The larger volume of oil extract on the glass

24

wool is thus recommended.

Fresh samples of the second oil extract were placed on glass wool

at different temperatures and the data filed as:-

50°C:

60 °C:

70°C:

80°C:

File 13

File 114

File 17

File 20

It is concluded that the solvent extraction method using oil is

not very useful because only one peak appears consistently in all

these chromatograms, namely that at about scan 300.

302o~ Microextractions

Simpler extractions on a much smaller scale were performed. This

technique was attempted because if successful, it would be a very

convenient method of extraction requiring little time and effort.

Furthermore, it would not involve the attrition of the more

volatile components which occurs in the concentration steps used

above.

A blank was run by injecting 5 ~l OCM using the following

temperature programme:

Initial temperature 40 °C


Initial time 5 min.


Final time 2 min.

25

Using the split mode CHROMATOGRAM 42 (File 100) was obtained.

Using the splitless mode for 25 s CHROMATOGRAM 43 (File OCM10l)

was obtained.

A mixture of 3 cm3 mahewu and 200 ~l OCM was shaken in a 5 cm3

syringe, allowed to separate and the OCM portion carefully

removed using a narrow J-tube connected to the syringe. 5 ~l of

the OCM extract was injected.

Using the split mode (File OCM102) was obtained.

Using the splitless mode for ·25 s CHROMATOGRAM 44 (File OCM103)

was obtained.

There is a difference between chromatograms 42 and 43 because

with the split, only 1/15th of the sample went on to the column.

Consequently, with the injection being splitless for a short

while, the whole sample is allowed to enter the column, the split

valve is opened and the sample chromatographed with a smaller

mass flow rate of carrier gas, seeing that most of the gas is

being split off. So the injection being splitless for 25 s

improves the situation.

comparing chromatograms 43 and 44, an extra peak appears at scan

254 on the sample chromatogram which is not on the blank, so this

micro-extraction method with a splitless mode for 25 s could be

used for the component producing this peak, but overall the

technique at present does not appear to be promising, mainly

because it appears to be so specific.

26

CHAPI'ER4

ANALYSIS

~ol IDENTIFICATION OF SOME COMPONENTS

The Library Search facility on the computer of the GC/MS was used

on all the files that were generated by the various runs

mentioned previously. A printout was obtained for each

significant peak. Each printout gave, inter alia, the three

compounds in its library whose mass spectra best fitted those of

the sample spectrum. From these were extracted those compounds

mentioned which appeared often. It was borne in mind that the

computer's library was limited. A list of these compounds

appears in Table 1, with the names of the files in which they

were found, the scan number of the peak, the rank given by the

computer, the purity of the match (which is an indication of the

closeness of the match) and the reconstructed ion chromatogram

count (RIC), which gave an indication of the quantity of the

compound present.

computer.

The names shown are those used by the

27

TABLE 401

SUBSTANCE CHROMAT SCAN RANK PURITY RIC

ACETIC ACID,ANHYDRIDE ETHEROI 131 3 736 9759

LPURGE3 320 1 836 3155

ACETIC ACID, HYDRAZIDE ETHER018 277 2 830 7439

ETHER078 391 5 594 88

ACETIC ACID,8UTYLESTER LPURGEIA 1152 3 645 4015

LPURGE2 542 2 938 5695

ACETIC ACID,ETHYLESTER ETHEROI 131 1 980 9759

ETHER018 277 1 920 7439

ETHER07A 269 1 975 177407

ETHER078 273 1 919 5639

LPURGEl 194 1 978

LPURGE2 202 1 989 83199

LPURGE3 320 2 932 3155

DEGOEDE5 97 1 979 202239

ACETIC ACID,ETHENYL ETHER018 277 5 776 7439

ESTER ETHER078 273 5 879 5639

20 173 3 868 9663

ACETIC ACID,HEXYLESTER LPURGEIA 1152 1 750 4015

ACETIC ACID,PENTYLESTER DEGOEDE43 554 1 940 46591

LPURGEI 798 1 930 29471

LPURGEIA 796 1 937 122239

LPURGE2 822 1 937 135679

LPURGE3 1134 2 932 1831

LPURGE3 1146 1 928 27487

LPURGE3 1167 2 951 11167

28

ACETIC ACID,PROPYLESTER ETHER01 131 2 788 9759

ETHER01B 220 4 793 7439

ETHER07A 269 3 741 177407

ETHER07B 273 3 812 5639

ACETIC ACID,TRIFLUORO- DEGOEDE43 415 2 807 1173

1,5-PENTANEDIYLESTER

ACETIC ACID, (1- ETHER07A 269 2 768 177407

METHYLETHOXY)- DEGOEDE5 97 2 769 202239

,ETHYLESTER

ACETIC ACID,2- LPURGE2 542 1 951 5695

METHYLPROPYLESTER

ACETIC ACID,2- HEXANE03 1751 1 930 1061

PHENYLETHYLESTER

BENZENE,1,4-DICHLORO LPURGE1A 1136 1 790 450

BENZENEETHANAMINE,N-(2- DEGOEDE43 826 1 863 741

PHENYLETHYL)-

,HYDROCHLORIDE

BENZENEETHANOL ETHER07A 1614 2 864 1022

DEGOEDE43 890 1 872 4223

29

BENZENE, ETHYL- HEXANE01 836 2 584 216

DEGOEDE43 517 1 766 1327

DEGOEDE43 532 3 943 18079

DEGOEDE43 573 3 939 23327

114 509 3 584 138

LPURGE1 757 1 932 1091

LPURGE1A 753 1 972 3251

LPURGE1A 778 3 914 9871

LPURGE1A 838 1 926 1811

LPURGE2 776 1 809 618

LPURGE2 801 1 934 1305

DEGOEDE5 438 1 998 1967

BENZENE,ETHENYL- LPURGE1 828 2 838 556

LPURGE1A 826 3 971 4231

LPURGE2 849 2 903 1169

LPURGE3 1189 1 781 305

BENZENE,1,2-DIETHYL LPURGE1 1251 2 919 4599

LPURGE1A 1257 2 868 158975

BENZENE,1,3-DIETHYL- LPURGE1 1251 3 917 4599

LPURGE1A 1257 3 864 158975

BENZENE,1,4-DIETHYL- LPURGE1 1251 1 937 4599

LPURGE1A 1257 1 894 158975

BENZENE,1-ETHENYL-2- DEGOEDE43 785 3 899 1711

METHYL-

BENZENE,ETHYLMETHYL- DEGOEDE43 785 5 882 1711

BENZENE, METHYL HEXANE01 556 5 699 178

HEXANE04 539 4 767 304

DEGOEDE43 890 4 751 4223

LPURGE2 521 1 971 5663

DEGOEDE5 228 1 970 4032

30

BENZENE,1,2-DIMETHYL HEXANE01 836 3 559 216

DEGOEDE43 517 2 688 1327

DEGOEDE43 532 1 957 18079

DEGOEDE43 573 1 947 23327

114 509 2 591 138

LPURGE1 757 3 932 1091

LPURGE1A 753 2 892 3251

LPURGE1A 778 1 926 9871

LPURGEIA 838 2 916 949

LPURGE2 801 2 934 1305

DEGOEDE5 438 2 964 1967

BENZENE, (l-METHYLETHYL)- DEGOEDE43 625 1 923 2767

DEGOEDE43 682 4 904 42047

DEGOEDE43 708 1 925 14975

LPURGE1A 1027 1 930 1315

LPURGE1A 1071 1 793 439

BENZENE,1-METHYL-2- DEGOEDE43 747 3 833 584

PROPYL- DEGOEDE43 806 2 941 3371

DEGOEDE43 826 2 841 741





DEGOEDE43 826 4 837 741

BENZENE, (1- DEGOEDE43 747 1 837 584

METHYLPROPYL)- DEGOEDE43 806 4 931 3371

DEGOEDE43 826 3 840 741

BENZENE, PROPYL- DEGOEDE43 669 1 966 10303

LPURGE1A 1008 1 914 783

BENZENE,l-PROPENYL- DEGOEDE43 785 4 888 1711

31

BENZENE,2-PROPENYL- DEGOEDE43 785 2 905 1711

BUTANOIC ACID,ETHYLESTER LPURGE2 612 1 914 1331

DEGOEDE5 306 1 983 3983

DEGOEDE5 741 5 833 2379

BUTANOIC ACID,2-ETHYL- DEGOEDE5 741 3 839 2379

l,2,3-PROPANETRIYLESTER

BUTANOIC ACID,3- DEGOEDE5 849 4 893 2033

METHYLBUTYLESTER DEGOEDE5 911 2 881 4615

BUTANOIC ACID,l- DEGOEDE5 306 5 846 3983

METHYLETHYLESTER

BUTANOIC ACID,2-METHYL- LPURGE1 1395 1 778 944

2-METHYLBUTYLESTER

2-BUTANOL,3-METHYL- DEGOEDE5 195 1 951 16383

1-BUTANOL,2-METHYL- LPURGE2 827 1 956 15935

,ACETATE LPURGE3 1167 3 881 11167

1-BUTANOL,3-METHYL- DEGOEDE43 554 2 928 46591

,ACETATE LPURGE1 798 2 908 29471

LPURGE1 796 2 921 122239

LPURGEl 892 1 904 8879

LPURGE2 822 2 923 135679

LPURGE2 827 2 906 15935

LPURGE3 1134 1 933 1831

LPURGE3 1146 2 915 27487

LPURGE3 1167 1 954 11167

CYCLOBUTANOL,2-ETHYL- 12 388 1 899 40191

20 355 1 903 273919

32

1,3,5-CYCLOHEPTATRIENE ETHER07A 1614 5 821 1022

HEXANE01 556 2 740 178

HEXANE04 539 3 758 304

LPURGE2 521 3 948 5663

DEGOEDE5 228 3 972 4023

1,3,5,7- LPURGE1 828 1 849 556

CYCLOOCTATETRAENE LPURGE1A 826 1 989 4231

LPURGE2 849 1 906 1169

LPURGE3 1189 2 779 811

CYCLOTRISILOXANE,HEXAMET 11 456 2 807 366

HYL-

FURAN,2,5-DIHYDRO- ETHER01. 154 3 845 773

ETHER018 541 4 857 1129

ETHER07A 306 4 809 16079

FURAN,2,3-DIHYDRO-4- ETHER07A 566 5 696 337

METHYL-

FURAN,4-METHYL-2-PROPYL- 12 732 2 696 495

FURAN,2-PENTYL- 12 680 1 909 1789

20 672 1 631 2699

LPURGEIA 1110 1 756 9343

DEGOEDE5 729 1 996 2099

1-HEPTANOL,6-METHYL- 2 646 5 798 526

2-HEPTANONE 20 522 1 944 41663

LPURGEIA 817 1 923 5311

2-HEPTANONE,4-METHYL- LPURGEl 1086 2 881 1759

4-HEPTANONE,3-METHYL- ETHER078 1833 4 789 475

HEXANE03 2159 2 812 785

HEXANOIC ACID DEGOEDE43 972 3 662 1943

33

•

HEXANOIC ACID,ETHYLESTER LPURGE1A 1118 1 790 770

LPURGE2 1140 1 921 1041

DEGOEDE5 741 1 987 2379

HEXANOIC ACID,2- LPURGE1A 1318 1 870 1335

PROPENYLESTER DEGOEDE5 887 1 961 3975

HEXANOIC ACID,2-METHYL- DEGOEDE5 146 4 892 8415

3-0XO-,ETHYLESTER

HEXANEDIOIC ACID,2,4- DEGOEDE5 1102 2 868 4951

DIMETHYL-,METHYLESTER

3-HEXANETHIOL,3-ETHYL- 4 1075 5 625 654

1-PENTANOL 2 296 3 853 1167

LPURGE1A 424 2 902 35071

DEGOEDE5 195 2 883 16383

LPURGE3 651 2 839 3875

1-PENTANOL,3-METHYL- 1 703 2 776 1605

12 695 4 703 1909

20 687 2 715 11007

2-PENTANONE 20 173 1 989 9663

2-PENTANONE,5-PHENYL- HEXANE03 1751 3 770 1061

4-PENTENAL,2-ETHYL 2 646 1 832 532

20 623 1 873 5671

1-PENTENE ETHER07B 574 3 815 695

HEXANE03 293 3 872 287231

4 300 3 846 14479

2-PENTENE,5- DEGOEDE 887 3 884 3975

(PENTYLOXY),(E)-

2-PENTEN-1-0L,2-METHYL- 20 544 4 787 13663

34

4-PENTEN-2-0L ETHER07B 75 3 746 227

3-PENTENOIC ACID,4- 4 99 4 835 30143

METHYL-

PHENOL,2,6-BIS(1,1- 20 1264 1 879 3179

DIMETHYLETHYL)-4-METHYL- 12 1252 1 833 1109

2 1216 1 814 1267

2 1276 1 727 597

4 1283 1 774 1063

PHENOL,2,6-BIS(1,1- 2 1216 2 680 1267

DIMETHYLETHYL)-4- 2 1276 3 536 597

METHYLCARBAMATE 4 1283 2 590 1063

12 1252 2 784 1109

20 1264 2 839 3179

PHENETHYLAMINE,N- DEGOEDE43 669 4 838 10303

BENZYL,P-CHLORO-

HYDROCHLORIDE

1-PROPANOL,2-METHYL ETHER07B 1764 1 741 57

2-PROPANOL ETHER01 62 4 841 47039

ETHER07B 75 2 802 227

2-PROPANONE HEXANE01 93 1 911 1845

4 99 1 968 30143

11 30 2 855 906

20 75 2 913 212735

20 522 4 811 41663

PROPANOIC ACID ,.ANHYDRIDE DEGOEDE43 246 2 727 1393

PROPANOIC DEGOEDE43 246 1 896 1393

ACID,ETHYLESTER LPURGE2 384 1 807 1169

2-PROPANONE,1-FLUORO- LPURGE3 320 4 804 3155

35

PROPANOIC ACID,2- ETHER01 62 5 832 47039

HYDROXY-,ETHYLESTER ETHER01B 76 4 671 465

1 93 3 856 33791

12 86 3 854 55167

PROPANOIC ACID,2- ETHER01 125 5 731 1349

HYDROXY-2-METHYL- ETHER07A 252 2 803 81535

ETHER07B 257 2 747 1625

PROPANOIC ACID,2-METHYL ETHER01B 220 4 784 4151

PROPANOIC ACID,2-METHYL- LPURGE2 612 2 782 1331

,ANHYDRIDE

PROPANOIC ACID,2-METHYL- LPURGE2 612 4 769 1331

,ETHYLESTER DEGOEDE5 306 2 933 3983

PROPANOIC ACID,2-METHYL- LPURGE1A 1354 1 802 873

,PENTYLESTER DEGOEDE5 911 1 978 4615

PROPANOIC ACID,2-METHYL- HEXANE03 1751 2 829 1061

,2-PHENYLETHYLESTER

From the above compounds were selected those with a rank of 1 and

a reasonable RIC count. These appear in Table 4.2, together with

some physical propertiesll•

TABLE 402

SUBSTANCE MELTING BOILING SOLUBILITY

POINT/oC POINT/oc

ACETIC ACID,ETHYLESTER -84 77 soluble

ACETIC ACID,PENTYLESTER -71 149 al, eth

36

BENZENE,ETHYL- -75 136 ai, eth

BENZENE,1,4-DIETHYL- -43 184 ai, eth, bz,

ac

BENZENE,METHYL- -95 111 ai, eth, ac,

bz

BENZENE, (l-METHYLETHYL)- -96 152 al, eth, ac,

bz

BENZENE, PROPYL- -100 159 al, eth, ac,

bz

BUTANOIC ACID,ETHYLESTER -101 121 al, eth

1,3,5,7-CYCLOTETRAENE -5 141 aL, eth, ac,

bz

2-HEPTANONE -36 151 ai, eth

PHENOL,2,6-BIS(1,1- 71 265 al, eth, bz,

DIMETHYLETHYL)-4-METHYL- chi

PROPANOIC ACID,ETHYLESTER -74 99 al, eth, ac

Considering the boiling points of the compounds, the properties

of the compounds of only those in the following table were

obtained.

37

TABLE .fi03

I SUBSTANCE I PROPERTIESI2 IACETIC ACID,ETHYLESTER Fruity odour; in artificial fruit

essences.

ACETIC ACID,PENTYLESTER

BENZENE, ETHYL- Irritant TLV 435

BENZENE,1,4-DIETHYL-

BENZENE, METHYL- Moderately toxic vapour.

BENZENE, (l-METHYLETHYL)- Toxic, irritant TLV 375

BENZENE, PROPYL- Moderately toxic

BUTANOIC ACID,ETHYLESTER

1,3, 5,7-CYCLOTETRAENE Highly flammable

FURAN,2-PENTYL Occurs in many foods including

coffee, potatoes, tomatoes, soya bean

oil - aroma. Probably results from

oxidation of unsaturated fatty acids

2-HEPTANONE Moderately toxic TLV 465

Alarm substance of ants. Flavour

ingredient. Light yellow oil.

PHENOL,2,6-BIS(1,1- Antioxidant. Moderately toxic.

DIMETHYLETHYL)-4-METHYL-

PROPANOIC ACID,ETHYLESTER Used as flavouring agent.

38

Considering the boiling points, other properties and availability

of the compounds, the following (with the names on the bottles

in parentheses) were purchased in order to confirm, or otherwise,

the presence of the substances in mahewu.

acetic acid,ethylester (ethyl acetate)

acetic acid,pentylester (pentyl acetate)

benzene,ethyl (ethyl benzene)

benzene,propyl (propyl benzene)

2-heptanone (pentyl methyl ketone)

propanoic acid,ethylester (ethyl propionate)

402 CONFIRMATION OF SUBSTANCES PRESENT IN THE HEADSPACE OF MAHEWU

This was performed on the GC.

To confirm whether the suspected sUbstances were present,

5.0 cm3 of each substance in turn was pipetted into a clean

sample tube, purged for 2 min. at room'temperature and introduced

into the GC, using the following temperature programme.

Initial temperature 50 °c

Final temperature 170 °c

Initial time 2 min.


final time 2 min.

39

The number of the chromatogram obtained for each sUbstance is

given in Table 4.4 together with the retention times for the main

peaks.

TABLE 4.4

SUBSTANCE CHROMATOGRAM RETENTION

NUMBER TIME/min.

ethyl acetate CHROMATOGRAM 45 2.4

ethyl propionate CHROMATOGRAM 46 3.0

ethyl benzene CHROMATOGRAM 47 5.4

pentyl methyl CHROMATOGRAM 48 6.2

ketone

propyl acetate CHROMATOGRAM 49 6.7 & 2.3

propyl benzene CHROMATOGRAM 50 7.6

A5.0 c m? sample of mahewu was purged for 10 min. and

chromatographed, producing CHROMATOGRAM 51.

This chromatogram had peaks corresponding with those from Table

4.4 at 2.4 min., 3.8 min. and 7.6 min. This indicates that ethyl

acetate, ethyl propionate and propyl benzene were present in the

headspace of mahewu. Peaks did not appear at 5.4 min., 6.2 min.

and 6.7 min. which suggests .that ethyl benzene, pentyl methyl

ketone and propyl acetate were not present.

40

The chromatograms of the headspace of mahewu show a large number

of peaks. What has been established here is the identity of only

three of these components of the headspace. The mass spectra of

the remainder were such that the computer c'ould not give

satisfactory matches with those in its library. Printouts of a

peak for each component identified are shown in the appendix as

MASS SPECTRA 1, 2 '3. In each case the result of the library

search on the mass spectrometer at the University of Natal,

Durban, is shown together with the mass spectrum of the sample

peak above, followed by the mass spectra of the three compounds

in the library whose mass spectra best match that of the sample.

Here propanoic acid, ethyl ester is ranked 2 whereas the original

rank of 1 was obtained on the mass spectrometer at Technikon

Natal.

403 QUANTITATION

This was performed on the Ge.

After a few investigative runs with different concentrations of

standards in the mahewu the following solutions were prepared to

determine the concentration of each of the suspected substances

in a mahewu sample using the method of standard addition.

Because the volumes added were small, no adjustment was made for

the volumes of solution.

100 ~l of each of the six substances in 100.0 cm3 mahewu

giving a mahewu solution 0.10% in each standard.

41




giving a mahewu solution 0.050% in each standard~



For each solution, 5.0 c m? was purged for 10 min. and

chromatographed, giving the following:

0.010% solution

0.075% solution

0.050% solution

0.025% solution

CHROMATOGRAM 52

CHROMATOGRAM 53

CHROMATOGRAM 54

CHROMATOGRAM 55

The heights of the peaks, measured from the interpolated

baselines for each standard substance is shown in Table 4.5.

TABLE 4.5

PEAK HEIGHT/mm

SUBSTANCE RETENTION % ADDED

TIME/min.0.025 0.050 0.075 0.10

ethyl acetate 2.4 26 16 50 92

ethyl propionate 3.8 28 21 48 62

ethyl benzene 5.4 - - - -propyl methyl 6.2 - - - -ketone

propyl acetate 6.7 - - - -propyl benzene 7.6 83 20 92 195

42

200 GRAPH I; Standard additiorrJl ru MahewlO oomponents

150

50

0;-----------,-----------,-----------,0.025 0.05 0.075

% added

Name of standardethyl acetateethyl propionatepropyl benzene

0.1

These graphs show that this method of standard addition is not

satisfactory for the determination of concentrations of these

SUbstances at these low concentrations. The concentrations of

those which give peaks could be said to be about 0.03%. The only

inference which can be made about the propyl benzene is that its

concentration.is less than 0.1%.

43

CHAPTER 5

DISCUSSION

The flavour component of foods total no more than 0.1%. When

analyzing the olfactory components there are a host of problems

associated with the analysis. There are usually many components

in varying concentrations and the components vary greatly in

their odoriferous thresholds. Also the response of the human

nose is completely different to that of a detector used in

chromatography. Often a component present in extremely small

concentration, thus producing a very small peak, has a profound

effect on the overall quality of the smell. The interaction of

olfactory components cannot be ignored and the aromas we

experience depend on the interaction of aromas of components

present.

With regard to sampling, cognisance must be taken of the fact

that the packaging can have an effect on the aroma. Often a

component is leached out of the material with which the food is

in contact. In the case of mahewu this is a carton. Sampling

the headspace above the manufacturers' tank obviates this

problem. This method of obtaining a sample does show

possibilities as shown by chromatogram 12. However, a suitable

method of removing the water from the headspace without removing

other constituents would have to be found. This would be the

44

best place to sample the headspace in order to detect at an early

stage any problems that arise in the production process. The

purge-and-trap method is suitable, both on the large tanks and

in the laboratory.

Whereas the addition of sodium chloride was used to attempt to

drive the volatiles out of the mahewu without much success,

various salts could be tried because they have different

effects .13•considering the solvent extraction techniques used here, none

seemed to be completely satisfactory for the purpose of

investigating the whole range of components which might be

extracted. However, they may be used for specific components.

Regarding chromatograph 21, it would be very difficult to resolve

any peak which is very close to the large solvent peak around

scan 100. These peaks would be due to components with low

boiling points, not being retained greatly by the column. One

of these components may well contribute greatly to the odour, so

another column or temperature programme or solvent would have to

be found which would separate this component's peak from the

solvent peak .. After scan 500 there appears a large number of

peaks which are not small judging by the total ion count of

6553590. These peaks could be resolved to a greater extent than

has already been done. These components must be of higher boiling

points, thus being retained longer on the column. These

45

compounds need careful consideration when assessing their

contribution to the aroma. The fact that a compound has a high

boiling point and low vapour pressure at room temperature does

not mean that it does not contribute greatly to -the aroma. If

certain of these compounds have a low threshold of detection by

olfactory receptors then they will have a profound effect on the

overall smell of mahewu.

• Of the solvents used for extraction only ether and the

sunflowerseed oil seemed to extract very little in the way of

volatile components. The others seemed to be effective. The

technique used to concentrate these extracts has caused the loss

of the more volatile components, especially the blow dry method.

This is shown by chromatograms 21 and 29, except that with the

blow dry method nothing appeared after the solvent peak. It

would be better to blow it not completely to dryness but only to

a certain volume. This may be difficult to reproduce as was the

case with using the rotary evaporator but the volumes can always

• be made up with solvent. The rotary evaporation technique of

concentration does mitigate mainly against the low boilers

because they would have high vapour pressures at ordinary

temperatures and greater rates of evaporation, under the vacuum

conditions.

46

Chromatogram 22, of hexane extract that had not been

concentrated, showed peaks of reasonable size, nowhere near the

solvent peak. For analysis of these components a concentration

technique is not required. This is highly desirable because of

possible losses as mentioned above.

•On the qualitative analysis side a great deal of work was done

but what was confirmed is very limited. It was established that

three substances, viz. ethyl acetate, ethyl propionate and propyl

benzene are constituents of the headspace of mahewu. It is

surprising that the butyl esters do not appear. It is also

noteworthy that the established components have relatively low

boiling points. This is a little surprising considering that the

later peaks in the chromatograms of mahewu are well resolved.

It does, however, depend on the mass spectrum obtained by the MS.

Quantitatively, what has been achieved is very limited. A rough

estimation of the concentrations of ethyl acetate and ethyl

propionate in mahewu is of the order of 0.03%, while it can only

be said that that of propyl benzene is less than 0.1%. Headspace

analysis by purge-and-trap is a way of analyzing qualitatively

for them but not quantitatively. This technique does not give

consistent enough results for it to be satisfactory. If the MS

is to be used as detector, long purges for 48 hours are necessary

for detection. The more sensitive flame ionization detector can

be used for purge times of only 10 minutes, but the electrometer

has to be set at a very sensitive value which mitigates against

accuracy when considering qu~ntitation.

•

47

In future work the nose could be introduced as a detector for

identification of odoriferous components of the effluent from

the column. 14 This approach enables the analyst to assess the

level of sensory importance attributable to given-regions of the

chromatogram. The "sniff port" can either be used after a non-

destructive detector, by a split-effluent technique or by

sequential separations. None of these provides the ideal

solution: the non-destructive detectors available cannot really

be used with high-resolution columns (i.e. not with capillary

columns); temperature variations affect the split ratio of a

stream splitter; sequential separations involve changing the

outlet of the capillary for successive runs, resulting in

variation of the retention time of a particular component. Also

capillary columns, which resolve mixtures well, are not sui table

for sniffing because of the small quantities of mixture

injected. 15

Work is being done on an "artificial olfactory sensor". 16 This

sensor consists of a set of piezoelectric sensors which

incorporate different hydrophobic polymer materials. This type

of sensor would obviate the need to rely on a subjective human

nose to perform the detection because it, like the nose, does not

separate compounds before responding to them. It therefore

offers grea~ possibilities.

It is clear from the present study, that mahewu, as with most

other food product, presents great challenges when it comes to

the analysis of volatile compounds present. Not only is the

dynamic range of compound concentration very great: extraction

48

is a problematic area, and detection/assessment of aroma

importance is still a major area in which progress has to be

made, before complete characterisation of the profiles of

volatiles of mahewu, and other foods, can be put on a scientific

footing which stands separate from the territory of the

sUbjective methods currently used in industry.

•

49

LIST OF REFERENCES

1. Schweigart, F., van Bergen, W.E.L., Wiechers S.G. and

de Wit, J.P. 1960. 'The Production of Mahewu'. C.S.I.R.

Research Report. No. 167• Torline, P.A. pers. com. National Food Research Institute,

P.o. Box 395, Pretoria, to D.N. Neethling. 1979.

3. Amato, I. 1988. ' The Anatomy of Flavour'. Analytical

2.

Chemistry, 60(15): 924A

4. Nitz, Sand Jtilich, E. 1984. 'Concentration and GC-MS

Analysis of Trace Volatiles by Adsorption-Desorption

Techniques'. Ed. P. Schreier. Analysis of Volatiles. Walter

de Gruyter & Co.: Berlin.

Curvers, J. Noy, Th., Cramers, C. and Rijks, J. 1984.5.

'Possibilities and Limitations of Dynamic Headspace

Sampling as a Pre-concentration Technique for Trace

Analysis of organics by Gas Chromatography'. J. of

Chromatography, 289 .171-182.

6. Van Deemter, J.J., Zuiderweg, F.J. and Klinkenberg, A.;

Chem. Eng. sci., 5, 271(1956)

7. Jennings, W.G., Rapp, A. 1983. Sample Preparation for Gas

Chromatographic Analysis. Dr. Alfred HtithigVerlag.

Heidelberg: 49.

50

•

8. Weast, R.C. 1984. CRC Rubber Handbook. CRC Press,lnc.

Boca Raton.

9. Jennings, w.G., Rapp, A. ibid.:49

10. Dupuy, H.P., Flick, G.J. Jr., St.Angelo, A.J.,

Sumrell, G. 1986. Analysis for Trace Amounts of Geosmin in

Water and Fish. JAOCS. 53(7): 905-908

11. Weast, R.C. ibid.

12. 1982. Dictionary of Organic compounds, 5th Edn, Chapman

and Hall, New York.

13. loffe, B.V. and Vitenberg, A.G. 1984. Head-Space Analysis

and Related Methods in Gas Chromatography. John wiley and

Sons: New York: 62

14. Parsons, W.A., Haarman and Reimer. 1992. 'Flavours and

Food'. Technology SA - Chemical, June. 5

15. Jennings, W. and Takeoka, G. 1984. 'State-of-the-Art Fused

Silica Capillary Gas Chromatography: Flavor Problem

Applications'. Ed. P. Schreier. Analysis of Volatiles.

Walter de Gruyter and Co.: Berlin.

16. Yokoyama, K. and Ebisawa, F. 1993. 'Detection and

Evaluation of Fragrances by Human Reactions Using a Sensor

Based on Adsorbate Detection'. Analytical Chemistry,

65(6): 673-677

•

51

APPENDIX

CHROMATOGRAM 1 to 55 and MASS SPECTRUM 1, 2 & 3 (all reduced to

85% of their original size) follow:

•

•

RI(002701501 W+W 1043

•

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CHROMATOGRAM 2

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CHROMATOGRAM 3lJ

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CHROMATOGRAM 13

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CHROMATOGRAM 14

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CHROMATOGRAM 15

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CHROMATOGRAM 17

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CHROMATOGRAM 47

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CHROMATOGRAM 48

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CHROMATOGRAM 54

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CHROMATOGRAM 55

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MASS SPECTRUM 1

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MASS SPECTRUM 2

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masters' diploma in technology (chemistry)i the development of a method for the analysis of...

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