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Progress report 1

WW0831 – Dr J Marais (ARC Infruitec-Nietvoorbij)

CFPA Canning Fruit Producers’ Assoc.

Submit to: Wiehahn Victor

PO Box 426 Paarl, 7620

Tel: +27 (0)21 872 1501

[email protected]

DFPT Deciduous Fruit Producers’ Trust

Submit to: Louise Liebenberg Suite 275, Postnet X5061

Stellenbosch, 7599 Tel: +27 (0)21 882 8470/1

[email protected]

DFTS Dried Fruit Technical Services

Submit to: Dappie Smit PO Box 426 Paarl, 7620

Tel: +27 (0)21 872 1501

[email protected]

Winetech

Submit to: Jan Booysen

PO Box 528 Suider-Paarl, 7624

Tel: +27 (0)21 807 3324

[email protected]

Indicate (X) client(s) to whom this progress report is submitted. Replace any of these with other relevant clients if required.

FINALFINALFINALFINAL REPORT REPORT REPORT REPORT

FOR FOR FOR FOR 2008200820082008/2009/2009/2009/2009

PROGRAMME & PROJECT LEADER INFORMATIONPROGRAMME & PROJECT LEADER INFORMATIONPROGRAMME & PROJECT LEADER INFORMATIONPROGRAMME & PROJECT LEADER INFORMATION

Programme leader

Project leader (ARC) Project leader (ARC)

Title, initials, surname Dr. J. Marais Dr. O.P.H. Augustyn Dr. I. Burger Present position Specialist Scientist Research Leader Senior Scientist Address ARC Infruitec-

Nietvoorbij ARC Infruitec-Nietvoorbij

ARC Infruitec-Nietvoorbij

Tel. / Cell no. (021) 809 3096 (021) 809 3010 (021) 809 3174 Fax (021) 809 3002 (021) 809 3002 (021) 809 3002 E-mail [email protected] [email protected] [email protected]

Project leader

(US-IWBT) Project leader (US-CHEM)

Project leader (UCT-Chem Eng)

Title, initials, surname Dr. P. van Rensburg Prof. A. Crouch Prof. S. Burton Present position Senior Lecturer Professor Associate Professor Address Institute for Wine

Biotechnology Department of Chemistry

Department of Chem. Engineering

Tel. / Cell no. (021) 809 7424 (021) 808 3535 (021) 650 2516 Fax (021) 808 3771 (021) 808 3342 (021) 650 5501 E-mail [email protected] [email protected] [email protected]

PROJECT INFORMATION

Project number WW 08/31

Project title Compilation of databases of aroma compounds in the wines of six cultivars

CFPA DFPT DFTS Winetech Production Technology

Industry programme

Other

Fruit kind(s) Wine grapes

Start date (dd/mm/yyyy) 01/01/06 End date (dd/mm/yyyy) 31/12/08

Progress report 2


FINAL SUMMARY OF RESEARCH REPORTFINAL SUMMARY OF RESEARCH REPORTFINAL SUMMARY OF RESEARCH REPORTFINAL SUMMARY OF RESEARCH REPORT

PROGRAMME & PROJECT LEADER INFORMATION

Programme leader Project leader (ARC)

Project leader (ARC)

Title, initials, surname Dr. J. Marais Dr. O.P.H. Augustyn Dr. I. Burger Institution ARC Infruitec-

Nietvoorbij ARC Infruitec-Nietvoorbij

ARC Infruitec-Nietvoorbij

Tel. / Cell no. (021) 809 3096 (021) 809 3010 (021) 809 3174 E-mail [email protected] [email protected] [email protected]

Project leader (US-IWBT)

Project leader (US-CHEM)

Project leader (UCT-Chem Eng)

Title, initials, surname Dr. P. van Rensburg Prof. A. Crouch Prof. S. Burton Institution University of

Stellenbosch University of Stellenbosch

University of Cape Town

Tel. / Cell no. (021) 809 7424 (021) 808 3535 (021) 650 2516 E-mail [email protected] [email protected] [email protected]

PROJECT INFORMATION

Project number WW 08/31

Project title Compilation of databases of aroma compounds in the wines of six cultivars

Fruit kind(s) Wine grapes

Start date (dd/mm/yyyy) 01/01/06 End date (dd/mm/yyyy) 31/12/08

Databases of aroma compounds were compiled for the wines of six cultivars, i.e. Sauvignon blanc, Chardonnay, Pinotage, Shiraz, Cabernet Sauvignon and Merlot. These wines were young, unwooded and representative of the various geographical regions in South Africa. The wines were analysed by four different methods at the ARC Infruitec-Nietvoorbij, The University of Stellenbosch and the University of Cape Town. Simultaneously, a new method for the analysis of wine volatiles was developed by the University of Stellenbosch for future use.

Progress report 3


FINAL REPORT

(Relevant publications may replace the final report) 1. Problem identification and objectives State the problem being addressed and the ultimate aim of the project.

The ability to guarantee authenticity and detect fraud in foods and beverages is becoming increasingly important internationally. The recent scandal concerning illegal flavour addition to Sauvignon blanc wines, and persistent rumours concerning similar actions in other cultivar wines, reflected badly on the local industry. A repetition could seriously damage the export drive and lead to a loss of consumer confidence locally as well. Against this background the South African Wine Industry was compelled to intervene and to launch an investigation into ways and means of detecting aroma fraud in wine. The ability to detect fraudulent addition of aroma constituents to wine can only be based on an extensive knowledge of what naturally occurs in wine, hence the call for a research project on the aroma profiles of a number of cultivar wines.

The aim of this project was to compile comprehensive databases (“finger-prints”) of aroma components in wines of the most important South African cultivars, originating from the various geographical regions. Simultaneously, new analytical methods will be investigated and developed for future analysis of wine volatiles. In addition, the composition of relevant flavour essences (commercial) and plant extracts will be examined to supply support data. The results of the research will assist authorities in future to detect fraud and prosecute offenders. 2. Workplan (materials & methods) List trial sites, treatments, experimental layout and statistical detail, sampling detail, cold storage and examination stages

and parameters.

The relevant methods are given in Addendum 2 (p. 11). 3. Results and discussion State results obtained and list any benefits to the industry. Include a short discussion if applicable to your results. This

final discussion must cover ALL accumulated results from the start of the project, but please limit it to essential information.

Milestone Achievement

1. Compilation of databases (“finger-prints”) of aroma components of six cultivars.

Objective has been achieved.

2. Development of a new method for future analysis of wine volatiles.

Objective has been achieved.

3. Analysis of commercial flavour essences and plant extracts.

Objective has been partially achieved. Tests on commercial flavour essences still in progress (University of Stellenbosch).

Unwooded, young wines of six cultivars (Sauvignon blanc, Chardonnay, Pinotage, Shiraz, Cabernet Sauvignon and Merlot) were sampled from the 2005, 2006 and 2007 Young Wine Show. Wines from exclusive regions, not normally represented at the Young Wine Show,

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were also collected. The wines were divided according to the regions given by Platter (2006). Fifteen regions were used, namely Constantia/Cape Point, Durbanville/Philadelphia, Elgin/Walker Bay, Franschhoek, Helderberg, Little Karoo, Northern Cape/Free State/North West, Olifants River, Paarl/Wellington, Robertson, Southern Cape, Stellenbosch, Swartland/Darling, Tulbagh and Worcester/Breedekloof/Villiersdorp. The number of wines, collected over the three vintages from the relevant regions, are shown in Addendum 1 (p. 9). The wines were distributed between the three participating centres, namely the ARC Infruitec-Nietvoorbij, the University of Stellenbosch and the University of Cape Town. The wines involved amounted to a total of 1005. With regard to the component-specific methods (methoxypyrazines and mercaptans), which focused on Sauvignon blanc, Cabernet Sauvignon and Merlot only, 555 wines were analysed. Two methods [Headspace extraction of volatiles (ARC) and Liquid/liquid extraction of volatiles (US)] were used to detect as many components as possible. In cases where components could not be identified, numbers were given. In the case of the other two methods [Headspace extraction of methoxypyrazines (ARC) and Liquid/liquid extraction of mercaptans (UCT)] only relevant impact components were detected. They were 2-isobutyl-3-methoxypyrazine (IBMP), 2-isopropyl-3-methoxypyrazine (IPMP) and 2-sec-butyl-3-methoxypyrazine (SBMP) in the case of the ARC, and 2-mercaptoethanol (ME), 2-mercaptoethyl acetate (MEA), 3-mercaptopropyl acetate (MPA) and 3-mercaptobutanol (MB) in the case of the UCT. They were also detected only in the wines of the cultivars in which they are expected to occur, namely Sauvignon blanc, Cabernet Sauvignon and Merlot. In cases where the concentration of the analyte was below the LOQ (limit of quantitation), it was given as zero. The results obtained in this project are presented as follows: The relevant methods used by each centre are given in Addendum 2 (p. 11). Typical chromatograms for each cultivar and each method are given in Addendum 3 (p. 18). The aroma profiles for each cultivar and each method are given in Addendum 4 (p. 47). Addendum 5 (p. 66) contains the aroma profiles for each cultivar and for each method, from two climatically-different regions. The two regions selected are a relatively cool region (Stellenbosch) and a relatively warm region (Robertson). Aroma profiles of cultivars from any of the other 15 regions, can be obtained on request. However, a few regions would be difficult to present, because not sufficient samples could be collected. When regional classification is attempted, successful separation could be affected by the fact that some wineries in warmer regions obtained grapes from cooler regions. This practise is allowed and these grapes are then used in their brands and bottled under their names. This is especially the case with Sauvignon blanc grapes from the South and West Coast regions. In such cases, it is recommended that the origin of the grapes is determined and taken into account where outliers are detected. Wines, spiked with specific plant extracts and flavourings, were analysed by each centre. These wines, prepared by the University of Stellenbosch, were the control wine, spiked individually with a green pepper extract, and green grass, green pepper and asparagus flavourings. In the case of the US, no differences between the control wines and their corresponding spiked ones could be observed. The method is either not sensitive or specific enough to detect the relevant impact components. Commercial flavour essences were also sent by the US to the KWV for analysis. So far no results could be obtained. In the case of the methoxypyrazines (ARC), marked increases in the concentration of IBMP were observed when the wines were spiked with green pepper extract and asparagus flavouring. No

Progress report 5


increases in IPMP and SBMP concentrations were observed. Furthermore no methoxypyrazine levels were enhanced when the wines were spiked with the green grass flavouring. With respect to the headspace volatiles (ARC), increases in the levels of some volatiles in all the spiked wines were observed. These increases were especially evident with small peaks at high retention times. Further studies will be needed to identify the relevant peaks and to determine whether these volatiles could be involved in possible future fraudulent activities. With respect to the mercaptans (UCT), no differences between the control wines and their corresponding spiked ones could be observed. This is expected, since the mercaptans are fermentation-produced compounds. In all cases, chromatograms showing differences in detected peaks, are available on request. A new method for the analysis of volatiles (Stir Bar Sorptive Extraction) was developed by the Chemistry Department of the University of Stellenbosch for future use by the wine industry. This method is presented in Addendum 6 (p. 99). Many factors may influence an aroma profile. In a study like this where hundreds of samples are collected, it is obvious that not all factors could be taken into account. Therefore, apart from the exclusion of wood contact, which could be obtained, the profile results presented are a function of different parameters. Variation in the levels of, for example, methoxypyrazines is large, because so many factors, such as climatic, viticultural and oenological parameters play a role. Nevertheless, one would expect that the variation in warm regions would be smaller, due to the limitation on methoxypyrazine levels as a result of too high temperatures. It is now the responsibility of each project leader to undertake further statistical analyses of his/her data and to present it as publications in high-rated journals, preferably the South African Journal of Enology and Viticulture. Discrimination between regions would be interesting, keeping in mind the factors discussed above. In all cases the broader project and Winetech should be acknowledged. Furthermore, it should now be decided by Winetech whether the databases will be maintained, and if so by which institution. An aspect which should be discussed thoroughly, is the fact that new technology is continually developed and present methods are not necessarily adequate for future use. In this respect, the new Stir Bar Sorptive Extraction method could be applied for future use in the wine industry. The question that arises is how the present methods can be replaced by new methods, concerning the maintenance of the databases. When adulteration would occur, more intensive studies would be needed to identify the compounds involved. The present aroma profiles and methodology will naturally serve as a good basis. 4. Accumulated outputs List ALL the outputs from the start of the project. The year of each output must also be indicated.

Technology developed

See Addenda 2 (p. 11) and 6 (p. 99). Human resources developed/trained

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Patents

Publications (popular, press releases, semi-scientific, scientific)

University of Stellenbosch Louw, L., Roux, K., Tredoux, A., Tomic, O., Naes, T., Nieuwoudt. H.H. & Van Rensburg, P., 2009. Characterisation of selected South African young cultivar wines using FTMIR spectroscopy, gas chromatography and multivariate data analysis. J. Agric. Food Chem. (submitted). Louw, L., 2007. M.Sc. Agric. Oenology. Chemical Characterization of South African Young Wines. Weldegergis, B. T., Tredoux, A. G. J. & Crouch, A. M., 2007. Application of a headspace sorptive extraction method for the analysis of volatile components in South African wines. J. Agric. Food Chem. 55, 8696–8702. Weldegergis, B. T. & Crouch, A. M., 2008. Analysis of volatiles in Pinotage wines by stir bar sorptive extraction and chemometric profiling. J. Agric. Food Chem. 56, 10225-10236. Weldegergis, B. T., De Villiers, A., McNeish, C., Seethapathy, S., Górecki, T. & Crouch, A. M., 2008. Characterization of volatile components of Pinotage wines using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-TOFMS). J. Agric. Food Chem. (submitted). Weldegergis, B. T., De Villiers, A., Kidd, M. & Crouch, A. M., 2009. Chemometric investigation of the volatile content in young South African wines. J. Food Chem.(submitted). Presentations/papers delivered

University of Stellenbosch Posters Weldegergis, B. T., Van Rensburg, P., Nieuwoudt, H. H. & Crouch, A. M. 2006. Development and validation of analytical methods for the analysis of volatile Compounds in South African wines. The 38th National Convention of the South African Chemical Institute (SACI), 3 – 8 December 2006, University of Kwazulu-Natal, Durban, South Africa. Weldegergis, B. T. & Crouch, A. M., 2008. Analysis of volatiles in Pinotage wines by stir bar sorptive extraction and chemometric profiling. The 32nd International Symposium on Capillary

Chromatography (ISCC) and 5th GC × GC Symposium, 28 – 30 May 2008, Riva Del Garda, Italy. Weldegergis, B. T., De Villiers, A., Seethapathy, S., McNeish, C., Górecki, T. & Crouch, A.

M., 2008. Characterization of South African Pinotage wines using GC × GC-TOFMS. 32nd

International Symposium on Capillary Chromatography (ISCC) and 5th GC × GC Symposium, 28 – 30 May 2008, Riva Del Garda, Italy.

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Presentations Louw, L., Nieuwoudt, H. H., Lambrechts, M. G., Swanepoel, M., Naes, T. & Van Rensburg, P., 2006. Profiles of the chemical composition of South African young wines. The 3rd International Enology & Viticulture Conference. Somerset West, South Africa. Louw, L., Nieuwoudt, H. H., Lambrechts, M. G., Swanepoel, M., Naes, T. & Van Rensburg, P., 2007. Profiles of the chemical composition of South African young wines. The 13th Australian Wine Industry Technical Conference. Adelaide, South Australia. Treurnicht, J., Nieuwoudt, H. H., Esbensen, K. E., Van Rensburg, P. & Watts, V. A., 2006. Authentication of Sauvignon blanc wines using a multivariate data analysis approach. The 3rd International Enology & Viticulture conference. Somerset West, South Africa. Weldegergis, B. T., Van Rensburg, P., Nieuwoudt, H. H. & Crouch, A. M., 2006. Headspace analysis of volatile compounds in South African wines by stir bar methods coupled to gas chromatography-mass spectrometry. 3rd International Enology & Viticulture Conference, 14 -17 November 2006, Somerset West, South Africa. Weldegergis, B. T., Tredoux, A. G. J. & Crouch, A. M., 2007. Application of headspace sorptive extraction for the analysis of volatile component in South African wines. 31st International Symposium on Capillary Chromatography & Electrophoresis (ISCCE), 28-30 November 2007, Albuquerque, NM, USA. Weldegergis, B. T., De Villiers, A., Seethapathy, S., McNeish, C., Górecki, T. & Crouch, . M.,

2008. Characterization of South African Pinotage wines using GC × GC-TOFMS. 32nd

International Symposium on Capillary Chromatography (ISCC) and 5th GC × GC Symposium, 28 – 30 May 2008, Riva Del Garda, Italy.

University of Cape Town Poster and presentations Chemrawn XII, Stellenbosch, 2007. Compilation of a Database of Mercaptans in Wine for the Detection of Adulterants. Bio-08 Conference, 2008, Grahamstown. Compilation of a Database of Mercaptans in Wine for the Detection of Adulterants. Cape Biotec Forum, 2008, Somerset West. Compilation of a Database of Mercaptans in Wine for the Detection of Adulterants.

© Agricultural Research Council, 2005. The content of this document may constitute valuable Intellectual Property and is confidential. It may not be read, copied, disclosed or used in any other manner by any person other than the addressee(s) and specifically not disclosed to another party submitting a proposal herein. Unauthorised use, disclosure or copying is strictly prohibited and unlawful.

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5. Total cost summary of project

ARC INFRUITEC-NIETVOORBIJ

Year Winetech THRIP Other TOTAL

Total cost in real terms for year 1 2005/06 544 136 566 345

1 110 481


953 933


927 450

TOTAL 1 478 994 1 512 870

2 991 864

UNIVERSITY OF STELLENBOSCH


Total cost in real terms for year 1 2005/06 244 000 183 000 427 000



Total cost in real terms for year 4 2008/09 40 000 0 40 000

TOTAL 850 000 667 750 1 517 750

UNIVERSITY OF CAPE TOWN





TOTAL 698 000 482 833 1 180 833


Progress report 9

ADDENDUM 1

Distribution of wines


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Table 1. Distribution of wines (2005, 2006 and 2007 vintages combined).

Cultivar R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10

R 11

R 12

R 13

R 14

R 15

Total

Sauvignon blanc

5 7 6 12 10 8 1 9 34 40 4 38 5 4 34 217 *

Chardonnay

2 2 1 2 1 13 4 9 17 40 --- 2 4 5 38 140

Pinotage

1 2 1 --- 3 10 5 9 26 13 --- 13 11 4 34 132

Shiraz

1 2 2 2 6 9 5 11 36 32 1 16 11 7 37 178

Cabernet Sauvignon

2 3 1 2 4 9 5 8 32 34 1 28 6 7 33 175 *

Merlot

1 4 1 --- 6 12 4 10 26 27 1 22 8 4 37 163 *

Total

1005 *555

R 1 = Constantia/Cape Point; R 2 = Durbanville/Philadelphia; R 3 = Elgin/Walker Bay; R 4 = Franschhoek: R 5 = Helderberg; R 6 = Little Karoo; R 7 = Northern Cape/Free State/North West; R 8 = Olifants River; R 9 = Paarl/Wellington; R 10 = Robertson; R 11 = Southern Cape; R 12 = Stellenbosch; R 13 = Swartland/Darling; R 14 = Tulbagh; R 15 = Worcester/Breedekloof/Villiersdorp.

*Wines analysed for methoxypyrazines and mercaptans only.


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ADDENDUM 2

Methods used by each centre


Progress report 12

ARC-INFRUITEC NIETVOORBIJ

Method: Headspace extraction of volatiles in wine (Solid-phase microextraction)

Reagents and Solvents Sodium chloride – puriss (Riedel-de Haën) Ethanol – Hipersolv, 99.7 – 100% (BDH) Fiber Solid-phase microextraction (SPME) manual holders and fibers were purchased from Supelco. All analyses were performed using a polydimethylsiloxane (PDMS) fiber with a film thickness of

100µm. New fibers were conditioned in the GC injection port at 250°C for 1 h. Fibers were

conditioned between injections at 250°C for 5 min with a split ratio of 5:1. The condition of the fiber was monitored by analysing a standard wine every 2 weeks (every 50 – 60 analyses). The standard wine was decanted into 100 ml PET bottles at the beginning of the study and stored at

4°C. The standard wine was analysed by SPME in the same manner as the samples. The %RSD of a selected set of peaks 28 peaks was monitored. Sampling

Wine samples were taken from cold storage (4°C) and allowed to reach room temperature before analysis. Glass bottles with a volume of 200 ml were used for sampling. Using an A grade volumetric flask, 100 ml wine was measured into the sampling bottle. A small magnetic stirring bar and 10 g of sodium chloride were added. The bottle was sealed with a double layer of aluminium foil and a screw cap with a hole just wide enough for the SPME needle to fit. The

bottle was placed in a water bath which was kept at a constant temperature of 30°C. The sample was stirred at a constant speed for 15 min to equilibrate. The SPME fiber was inserted into the headspace by piercing the aluminium foil with the needle. The headspace was sampled

for 60 min with constant stirring at 30°C. The fiber was subsequently desorbed in the GC injector for 5 min with a split ratio of 10:1. Chromatographic conditions The samples were analysed using two Varian 3800 gas chromatographs with FID detectors. Each GC was dedicated to 3 cultivars. The gas chromatographs were equipped with Supelcowax-10 fused silica columns (60 m x 0.32 mm i.d.) with a film thickness of 0.25 µm

(Supelco). The injectors and detectors were kept at 250°C. A starting temperature of 60°C was

held constant for 5 min then increased by 2°C/min to 190°C and held for 30 min. A final ramp of

15°C/min increased the temperature to 240°C, where it was held for 37 min. The carrier gas was helium at a constant flow rate of 2.5 ml/min. The split ratio was 10:1 at the time of injection for 5 min and thereafter 5:1 for the rest of the run.

The fiber was conditioned for the next extraction, during the GC run by inserting it into the GC injector 75 min after the start of the run and removing it after 5 min.

The areas of the peaks were normalised with respect to peak no. 23 for Sauvignon blanc, peak no. 16 for Chardonnay, peak no. 18 for Pinotage, peak no. 16 for Shiraz, peak no. 24 for Cabernet Sauvignon and peak no. 17 for Merlot, respectively, as 100% (see Addendum 3).


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Method: Headspace extraction of methoxypyrazines in wine Reagents and Solutions

Sodium chloride was purchased from Riedel-de Haën. Ethanol (Chromasolv), and standard reference compounds of 2-isobutyl-3-methoxypyrazine (IBMP) (99% pure), 2-sec-butyl-3-methoxypyrazine (SBMP) (99% pure), and 2-isopropyl-3-methoxypyrazine (IPMP) (97% pure) were purchased from Sigma-Aldrich. The internal standard, 3-isopropyl-2-ethoxypyrazine (IPEP) was purchased from Pyrazine Specialties (Atlanta, GA, USA). Individual stock standard solutions of each of the pyrazines were prepared in ethanol and stored in darkness at -4˚C. Working solutions of each of the three methoxypyrazines (2.5 ng/ml), and of IPEP (250 ng/ml), respectively, were prepared in ethanol from the stock standard solutions. A global solution containing 2.5 ng/ml of each of the three methoxypyrazines in ethanol was prepared from the stock standard solutions for calibration purposes. The working and global solutions were stored in darkness at 4˚C. HS-SPME Procedure Final procedure for wines: 50 ml of wine was spiked with 20 µl of IPEP (100 ng/L). A volume of 10 ml of this solution was pipetteted into a 20 ml round bottomed flask, containing a Teflon stirrer bar. Concentrated HCl was used to adjust the pH to 0.50 and the volume of the sample was reduced to 50% by the evaporation of ethanol and other volatile components under reduced pressure at room temperature. The pH of the resulting solution was adjusted to pH 7 with a sodium hydroxide solution (20% and 5%). Sodium chloride (3 g) was added and the flask was closed with an adapter sealed with a PTFE-faced silicone septum. The sample was equilibrated for 15 min, before a 90 min extraction in the headspace of the flask using a Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) 50/30 µm fibre (Supelco). During equilibration and extraction, the sample was agitated at 700 rpm in an isothermal water bath at 30˚C. Analytes were thermally desorbed in the GC injector port at 250˚C for 5 min. Fibres were conditioned at 270˚C for 1 h before use and the 5 min desorption time was sufficient to prevent carry over between consecutive sample runs. Chromatographic Conditions The HS-SPME-GC/MS analysis was performed with a Finnigan Mat GCQ gas chromatograph – ion trap mass spectrometer equipped with a Zebron ZB-Wax-Plus fused silica column (30 m x 0.25 mm i.d., 0.25 µm film thickness) from Separations. Helium, at a constant velocity of 28.5 cm/sec, was used as carrier gas. Oven temperature was programmed as follows: 40°C for 5 min, heated at 1°C/min to 75°C and kept for 20 min and finally raised to 220°C at 10˚C/min and held for 10 min. Injections were made in the splitless mode for 1 min and then split was set at 10 ml/min. The injector temperature was kept at 250°C. The transfer line and ion trap temperatures were set at 230°C and 150°C, respectively. Mass spectra were acquired using electron impact ionisation (70 eV) and a scan rate of 1 s/scan. Full spectral information in a mass-to-charge (m/z) range 30 to 210 was collected. Ion m/z 124 was used for the quantification of IBMP, m/z 137 for IPMP, m/z 138 for SBMP and ion m/z 151 for the quantification of IPEP. The repeatability of the method was evaluated after 6 consecutive analyses of a Chenin blanc wine spiked with 10 ng/L of each of the three methoxypyrazines. Repeatability in terms of RSD was acceptable, ranging from 3.8 to 8.9 % for the different analytes.


Progress report 14

Calibration curves for the determination of methoxypyrazine in Sauvignon blanc wine were constructed using a Chenin blanc wine to which concentrations between 1 and 100 ng/L of the global solution were added in addition to the internal standard. A Shiraz wine was used for calibration to determine the methoxypyrazines in Merlot and Cabernet Sauvignon wines. Each concentration level was analyzed with both fibres. Each respective methoxypyrazine peak in relation to the internal standard peak was linearly correlated with the concentration of that specific methoxypyrazine in the standard. The method shows a satisfactory linearity with the coefficients of determination (R2) ranging from 0.9931 to 0.9999. Regression equations were used to quantify the methoxypyrazines in all samples. The limit of quantification of IBMP is 1 ng/L and that of both IPMP and SBMP, 2 ng/L, which is at the odour detection thresholds of these compounds in white wine. The detection limits of these compounds are below the odour detection thresholds. The recovery of the HS-SPME procedure was determined by the standard addition technique. The global solution was added to two different Sauvignon blanc wines at 2 concentration levels (5 and 25 ng/L). Samples from each level were extracted in triplicate. Recoveries were satisfactory for the concentration range analyzed.


Progress report 15


Method: Liquid/Liquid extraction of volatiles in wine

Chemicles, standards and wine matrix stimulant. Chemicals and standards. Ethyl acetate and isoamyl acetate were purchased from Riedel de Haën (Seelze, Germany). Methanol, hexanol, acetic acid and 2-phenylethanol as well as diethyl ether, ethanol and Na2SO4 were purchased from Merck (Darmstadt, Germany). Ethyl butyrate, propanol, isobutanol, butanol, hexyl acetate, ethyl lactate, propionic acid, iso-butyric acid butyric acid, iso-valeric acid, diethyl succinate, valeric acid, 2-phenylethyl acetate, 4-methyl-2-pentanol and hexane were purchased from Fluka (Buchs, Switzerland). Hexanoic acid, octanoic acid, isoamyl alcohol, ethyl octanoate and ethyl decanoate were purchased from Aldrich (Steinheim, Germany). Decanoic acid and ethyl hexanoate were purchased from Sigma (St. Louis, USA). Wine matrix simulant. The internal standard and volatile standards were dissolved in a wine simulant consisting of 12 %v/v ethanol and 2.5 g/L tartaric acid Merck (Darmstadt, Germany) in de-ionized water from a MilliQ water purifying system from Millipore (Billeric, MA, USA), pH adjusted to 3.5 with 0.1M NaOH Merck (Darmstadt, Germany). Liquid-liquid extraction procedure. Five mL of wine with internal standard, 4-methyl-2-pentanol, (100 µL of 0.5 mg/L solution in wine simulant) was extracted with 1 mL of diethyl ether by sonicating the ether/wine mixture for five minutes. The wine/ether mixture was then centrifuged at 3600 g for 3 minutes. The ether layer was removed and dried on Na2SO4. Each extract was injected into the GC-FID in triplicate. This method has been validated in terms of selectivity, linearity, limits of detection and quantification, recovery, robustness and repeatability. However, the results of the validation will not be discussed in this paper as it is beyond the scope of the article. Gas chromatographic conditions. A J & W DB-FFAP capillary GC column (Agilent, Little Falls,

Wilmington, USA) with dimensions 60 m length × 0.32 mm i.d. × 0.5 µm f.t and a Hewlett Packard 6890 Plus GC (Little Falls, USA) equipped with a split\splitless injector and an FID detector was used. The initial oven temperature was 33°C for 17 minutes after which the temperature was increased by 12°C/minute to 240°C, at which it was held for 5 minutes. Three microlitres of the diethyl ether extract was injected at 200°C. The split ratio was 15:1 and the split flow rate 49.5 ml/minute. The column flow rate was 3.3 mL/minute using hydrogen as a carrier gas. The detector temperature was 250°C. After each sample run, a post run of 5 minutes at oven temperature 240 °C, with a column flow of 6 mL/min cleaned the column from high boiling contaminants.


Progress report 16


Method: Liquid/Liquid extraction of mercaptans in wine The method used was a modified method developed by Tominaga et al., 2006 for the extraction of mercaptans from wine. Preparation of the resin: 20g of resin Dowex 1X2 Cl- form (Sigma, 44290) was reactivated by suspending it with a 0.1M hydrochloric acid solution. The resin was then rinsed with distilled water until a pH of between 5 and 6 was achieved. The resin was then loaded into a glass column (50mL glass column with 1.5cm diameter and removable tap) such that the stacked resin length is 30cm. Fresh resin was used for each sample. Both ends of the glass column were attached to a peristaltic pump.

Preparation of wine sample: 10mL of a p-hydroxymercuribenzoate (Merck, 8203080005) solution (2mM in 0.1M Tris-HCl, pH 7) was added to 50mL of wine (adjusted to pH 7) containing 10µL propyl thioacetate (Industrial Analytical, B21875) as an internal standard and 1mL α-lipoic acid (1µM) (Merck, 437692) as an antioxidant. The solution was stirred for five minutes using a magnetic stirrer. Extraction of sample: Through the top of the column, the wine solution is pumped through and then the resin is rinsed with 50mL sodium acetate buffer (0.1M, pH 6). The column is then inverted and the volatile thiols are released from the thiol-pHMB conjugate fixed on the column by eluting it using a cysteine solution (1.5g/100mL buffer adjusted to pH 6). This is also done through the end of the column that is now on top. Collect the eluate containing the volatile and extract with 1mL ethyl acetate and 10mL dichloromethane twice in a separating funnel. Collect the organic phases, dry on anhydrous sodium sulphate and then under nitrogen stream to approximately 25µL. The extract was stored at -20°C under nitrogen and protected from light to prevent degradation, primarily due to oxidation of the sulfhydryl groups. GC-FID analyses: A Thermo Finnigan gas chromatographer equipped with FID detection. Inject 5µL of the extract on a CP Wax 58/FFAP FS (50m x 0.32mm I.D. x 0.2µM). The operating temperatures: 2°C/min from 40°C to 240°C.; injector temperature, 250°C; detector temperature, 250°C. Nitrogen gas flow rate 4.8mL/min, split ratio 3. The extract was injected within 24 hours of extraction. Reproducibility of extraction method and calibration curves: Calibration curves were done to validate the extraction method. The wine chosen for calibration was a wine that had small peaks at the same retention times as that of the standard compounds. The calibration of the standard compounds was therefore corrected by subtracting the areas of the peaks originally in the wine from the spiked sample area. The area of the corrected peak/ area of internal standard were used to generate the calibration curve. The wines chosen for the calibration curve were prepared as described above but with between 0-10µg of the standard compounds added. Standard compounds chosen were 2-mercaptoethanol (ME); 3-mercaptohexanol (MH); 3-mercapto-3-methylbutan-1-ol (MB); 3-mercaptopropyl acetate (MPA); 2-mercaptoethyl acetate (MEA); 4-mercapto-4-methylpentan-2-one (MMP); 2-furanmethanethiol


Progress report 17

(FM); 2-methyl-3-furanthiol (2M3F) as (1) these were the compounds associated with the characteristic aroma of wine and (2) availability. The four latter compounds were not quantified as MEA and 2M3F were not detected in any of the wines, while MMP could not be detected using FID detection and FM continued to co-elute with acetic acid even after all efforts to separate the peaks.


Progress report 18

ADDENDUM 3

Typical chromatograms for each cultivar and each method


Progress report 19


Method: Headspace extraction of volatiles


Progress report 20

3432302826242220181614121086

3,000

2,900

2,800

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

1

2

3

4

5 6 7 8

9

10

11 12

13

14

15

16

17

18

1920 21

22

RT [min]

GC2_2008_Analysis_114.DATAµV

58565452504846444240383634

3,000

2,900

2,800

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

23 24

25

26 2728

29 30

31

32

33

35

36

37 38

39

40

41

42

43

44

RT [min]


Fig.1. Chromatogram of a typical Sauvignon blanc wine (1 = isobutyl acetate; 2 – 3 = unknown; 4 = isobutanol; 5 = isoamyl acetate; 6 = isoamyl alcohol; 7 = ethyl hexanoate; 8 = hexyl acetate; 9 – 12 = unknown; 13 = 1-hexanol; 14 = unknown;

15 = ethyl octanoate; 16 – 22 = unknown; 23 = ethyl decanoate; 24 = unknown; 25 = diethyl succinate; 26 – 30 = unknown; 31 = 2-phenylethyl acetate; 32 – 35 = unknown; 36 = 2-phenylethyl alcohol; 37 – 44 = unknown).


Progress report 21

363432302826242220181614121086

3,000

2,900

2,800

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

1

2

3 4 5 6

7

8

9

10

11

12

13

1415

RT [min]


585654525048464442403836

3,000

2,900

2,800

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

16 17

18

19

20

21

22

23

24

25 26

27

28

29

31

32

36

37

RT [min]


Fig. 2. Chromatogram of a typical Chardonnay wine (1 = isobutyl acetate; 2 = isobutanol;

3 = isoamyl acetate; 4 = isoamyl alcohol; 5 = ethyl hexanoate; 6 = hexyl acetate; 7 = 1-hexanol; 8 – 9 = unknown; 10 = ethyl octanoate; 11 – 15 = unknown; 16 = ethyl decanoate; 17 = unknown; 18 = diethyl succinate; 19 – 22 = unknown; 23 = 2-phenylethyl acetate; 24 – 27 = unknown; 28 = 2-phenylethyl alcohol; 29 – 37 = unknown).


Progress report 22

3432302826242220181614121086

3,000

2,900

2,800

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

1

2

3 8 9

10

12

13

14

15

16

17

RT [min]


727068666462605856545250484644424038

3,000

2,900

2,800

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

18

19

21

22

23

24

26

27

29

30

31

32

33

34

35

RT [min]


Fig. 3. Chromatogram of a typical Pinotage wine (1 = isobutyl acetate; 2 = isobutanol;

3 = isoamyl acetate; 8 = isoamyl alcohol; 9 = ethyl hexanoate; 10 = hexyl acetate; 12 = unknown; 13 = 1-hexanol; 14 = unknown; 15 = ethyl octanoate; 16 – 17 = unknown; 18 = ethyl decanoate; 19 – 23 = unknown; 24 = 2-phenylethyl acetate; 26 – 27 = unknown; 29 = 2-phenylethyl alcohol; 30 – 35 = unknown).


Progress report 23

3432302826242220181614121086

3,000

2,900

2,800

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

123

4

5 6 7

8

9

10

11

12

13

1415

RT [min]


6866646260585654525048464442403836

3,000

2,900

2,800

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

16

17

18

19

21

22

23

24

25

26

27

28

29

31

32

34

35

36 37

38

RT [min]


Fig. 4. Chromatogram of a typical Shiraz wine (1 = isobutyl acetate; 2 – 3 = unknown;

4 = isobutanol; 5 = isoamyl acetate; 6 = isoamyl alcohol; 7 = ethyl hexanoate; 8 = hexyl acetate; 9 – 10 = unknown; 11 = 1-hexanol; 12 = unknown; 13 = ethyl octanoate; 14 – 15 = unknown; 16 = ethyl decanoate; 17 – 29 = unknown; 31= 2-phenylethyl alcohol; 32 – 38 = unknown).


Progress report 24

363432302826242220181614121086

3,000

2,900

2,800

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

1 2

3

4 5

7

8

10 11

12

13

14

15

16

17

18

19

20 21

22 23

RT [min]

Aroma_GC1_Analysis_162.DATAµV

706866646260585654525048464442403836

3,000

2,900

2,800

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

43

44

45

47

48

49

50

51

52

53

RT [min]

Aroma_GC1_Analysis_162.DATAµV

Fig. 5. Chromatogram of a typical Cabernet Sauvignon wine (1 = isobutyl acetate;

2 – 3 = unknown; 4 = isobutanol; 5 = isoamyl acetate; 7 – 8 = unknown; 10 = isoamyl alcohol; 11 = ethyl hexanoate; 12 = hexyl acetate; 13 – 14 = unknown; 15 = 1-hexanol; 16 = unknown; 17 = ethyl octanoate; 18 – 23 = unknown; 24 = ethyl decanoate; 25 – 38 = unknown; 39 = 2-phenylethyl alcohol; 40 – 53 = unknown).


Progress report 25

3432302826242220181614121086

3,000

2,900

2,800

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

1

23

4

5 6 7

8

9

10

11

12

13

14

15

16

RT [min]


58565452504846444240383634

3,000

2,900

2,800

2,700

2,600

2,500

2,400

2,300

2,200

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

17

18

19

20

21

22

24

25

2627

28

29

31

32

33

34

35

36

3738

39

RT [min]


Fig. 6. Chromatogram of a typical Merlot wine (1 = isobutyl acetate; 2 – 3 = unknown; 4 = isobutanol; 5 = isoamyl acetate; 6 = isoamyl alcohol; 7 = ethyl hexanoate; 8 = hexyl acetate; 9 – 10 = unknown; 11 = 1-hexanol; 12 – 13 = unknown; 14 = ethyl octanoate; 15 – 16 = unknown; 17 = ethyl decanoate; 18 – 39 = unknown).


Progress report 26


Method: Headspace extraction of methoxypyrazines


Progress report 27


Progress report 28


Progress report 29


Progress report 30


Method: Liquid/Liquid extraction of volatiles


Progress report 31

Fig. 1. Chromatogram of a typical Sauvignon blanc wine (See close-up below).

1

min6 8 10 12 14 16 18 20 22

pA

0

50

100

150

200

250

FID1 A , (DATA20~1\KLAARI~1\LE000013.D )

1

2 3

5

4

6

7

8

Peaks 1-8: Ethyl Acetate, Methanol, Ethyl Butyrate, Propanol, Isobutanol,

Isoamyl acetate, Butanol, 4-Methyl-2-pentanol (IS)

10 15 20 25 30

pA

50

100

150

200

250

FID1 A, (DATA20~1\KLAARI~1\LE000013.D)

Sauvignon blanc


Progress report 32

min23 23.5 24 24.5 25 25.5 26 26.5 27

pA

20

30

40

50

60


9

10

1112

13

14

15

16

1718

19

Peaks 9-19: Isoamyl alcohol, Ethyl hexanoate, Hexyl acetate, Ethyl lactate, Hexanol, Ethyl

Caprylate, Acetic acid, Unknown 4, Propionic acid, Isobutyric acid, Unknown 5.

min28 29 30 31 32 33

pA

20

40

60

80

100

120

140


2022

2324

25

14

26

21

27

2829

Peaks 20-29: Butyric acid, Ethyl Caprate, Iso-valeric acid, Dietyl Succinate, 2-Phenyl acetate,

Hexanoic acid, 2-Phenylethanol, Octanoic acid, Decanoic acid, Unknown 15


Progress report 33

Chardonnay

min10 15 20 25 30

pA

0

100

200

300

400

500

600

FID1 A, (DATA20~1\KLAARI~1\LL000261.D)

Fig. 2. Chromatogram of a typical Chardonnay wine (See close-up below).

1

min6 8 10 12 14 16 18 20 22

pA

0

50

100

150

200

250


1

2 3

5

4

6

7

8

Peaks 1-8: Ethyl Acetate, Methanol, Ethyl Butyrate, Propanol, Isobutanol, Isoamyl acetate, Butanol,

4-Methyl-2-pentanol (IS)


Progress report 34

23 24 25 26 27 28

pA

20

40

60

80

100

120

140

160

180


9

10

11 12

1314

15

161718

19

20 21 22

Peaks 9-22: Isoamyl alcohol, Ethyl hexanoate, Hexyl acetate, Ethyl lactate, Hexanol, Ethyl

Caprylate, Acetic acid, Unknown 4, Propionic acid, Isobutyric acid, Butyric acid, Ethyl Caprate, Iso-

valeric acid, Diethyl Succinate

min29 30 31 32 33

pA

20

25

30

35

40


2324 25

26

27

29

3031

28

32

3334

35

36

Peaks 23-36: Unknown 6, Valeric acid, Unknown 7, Unknown 8, 2-Phenyl acetate, Hexanoic acid, 2-

Phenylethanol, Unknown 11, Octanoic acid, Decanoic acid, Unknown 15, Unknown 16.


Progress report 35

Pinotage

min10 15 20 25 30

pA

0

50

100

150

200

250

300

350

400

FID1 A , (DATA20~1\KLAARI~1\LL000404.D)

Fig. 3. Chromatogram of a typical Pinotage wine (See close-up below).

min6 8 10 12 14 16 18 20 22

pA

20

40

60

80

100

120

140


1

2

3

4

5

6

7

Peaks 1-7: Ethyl Acetate, Methanol, Propanol, Isobutanol, Isoamyl acetate, Butanol, 4-Methyl-2-

pentanol (IS)


Progress report 36

min23 24 25 26 27 28

pA

20

25

30

35

40

45

50

55


9

10

11

12

13

14

15

17

19

20

8

1618

Peaks 8-20: Isoamyl alcohol, Ethyl hexanoate, Hexyl acetate, Ethyl lactate, Hexanol, E thyl

Caprylate, Acetic acid, Unknown 4, Propionic acid, Isobutyric acid, Butyric acid, Ethyl Caprate, Iso-

valeric acid, Diethyl Succinate

min29 30 31 32 33

pA

20

30

40

50

60

70

80


2223

2425

2930

26

31

32

33

34

35

Peaks 23-35: Unknown 6, Unknown 7, Unknown 8, 2-Phenyl acetate, Hexanoic acid, Unknown 9,

Unknown 10, 2-Phenylethanol, Octanoic acid, Unknown 13, Decanoic acid, Unknown 14, Unknown

15, Unknown 16.

27

28


Progress report 37

Shiraz

min10 15 20 25 30

pA

0

100

200

300

400

500

600

700

800

900


Fig. 4. Chromatogram of a typical Shiraz wine (See close-up below).

min8 10 12 14 16 18 20 22

pA

50

100

150

200

250

300

350


1

2 35 6

7

8

Peaks 1-9: Ethyl Acetate, Methanol, Propanol, Isobutanol, Isoamyl acetate, Butanol,

4-Methyl-2-pentanol (IS ), Isoamylalcohol, Ethyl hexanoate

9

4


Progress report 38

min24 25 26 27 28

pA

20

30

40

50

60

70


10

1112

13

1415

17

20

18

Peaks 10-22: Ethyl hexanoate, Unknown 1, Unknown 2, Unknown 3, Ethyl lactate, Hexanol, Ethyl

Caprylate, Acetic acid, Unknown 4, Propionic acid, Isobutyric acid, Unknown 5, Butyric acid, Ethyl

Caprate, Iso-valeric acid, Diethyl Succinate

1621

22

19

23

24

25

min29 30 31 32 33 34

pA

25

30

35

40

45

50

55

60

65


30

31

26

32

332728

3638

39

40

41

Peaks 26-41: Unknown 6, Valeric acid, Unknown 7, Unknown 8, 2-Phenyl acetate, Hexanoic acid,

Unknown 9, Unknown 10, 2-Phenylethanol, Octanoic acid, Unknown 13, Decanoic acid, Unknown

14, Unknown 15, Unknown 16.

29

34

37


Progress report 39

Cabernet

min10 15 20 25 30

pA

0

100

200

300

400

500

600


Fig. 5. Chromatogram of a typical Cabernet Sauvignon wine (See close-up below).

8 10 12 14 16 18 20 22

pA

25

50

75

100

125

150

175

200


1

2 34

5

6

7

8

Peaks 1-9: Ethyl Acetate, Methanol, Ethyl Butyrate,Propanol, Isobutanol, Isoamyl acetate, Butanol,

4-Methyl-2-pentanol (IS), Isoamylalcohol

9


Progress report 40

min23 .5 24 24.5 25 25.5 26 26.5 27 27.5

pA

20

30

40

50

60


10

1112

13

1415

17

1819

Peaks 10-21: Ethyl hexanoate, Unknown 1, Unknown 2, Unknown 3, Ethyl lactate, Hexanol, Ethyl

Caprylate, Acetic acid, Unknown 4, Propionic acid, Isobutyric acid, Unknown 5.

15

20

21

min28 29 30 31 32 33 34

pA

20

30

40

50

60

70

80

90


22

23

24

28

29

30

25

31

3233

34

3526 27

36

37

38

39 40

41

42

Peaks 23-36: Butyric acid, Ethyl Caprate, Iso-valeric acid, Diethyl succinate, Unknown 6, Valeric

acid, Unknown 7, Unknown 8, 2-Phenyl acetate, Hexanoic acid, Unknown 9, Unknown 10, 2-

Phenylethanol, Unknown 11, Unknown 12, Octanoic acid, Unknown 13, Decanoic acid, Unknown



Progress report 41

Merlot

min10 15 20 25 30

pA

0

100

200

300

400

FID1 A, (DATA20~1\SIG10937.D)

Fig. 6. Chromatogram of a typical Merlot wine (See close-up below).

min8 10 12 14 16 18 20 22

pA

10

20

30

40

50

60

70

80

90


1

23

5

6

7

8

Peaks 1-8: Ethyl Acetate, Methanol, Ethyl Butyrate, Propanol, Isobutanol, Isoamyl acetate, Butanol,

4-Methyl-2-pentanol (IS)

4


Progress report 42

23 24 25 26 27 28

pA

20

30

40

50

60

70

80


1011

1213

14

15

1720

18

Peaks 9-26: Isoamyl alcohol, Ethyl hexanoate, Hexyl acetate, Unknown 1, Unknown 2, Unknown 3, Ethyl lactate, Hexanol, Ethyl Caprylate, Acetic acid, Unknown 4, Propionic acid, Isobutyric acid,

Unknown 5, Butyric acid, Ethyl Caprate, Iso-valeric acid, Diethyl Succinate

16

21

221923

24

25

9

25

min29 30 31 32 33 34

pA

20

30

40

50

60

70


30

31

32

33

35

27

3839

40

41

Peaks 26-41: Unknown 6, Valeric acid, Unknown 7, Unknown 8, 2-Phenyl acetate, Hexanoic acid,

Unknown 9, Unknown 10, 2-Phenylethanol, Octanoic acid, Unknown 13, Decanoic acid, Unknown


29

3437

28

36


Progress report 43


Method: Liquid/Liquid extraction of mercaptans


Progress report 44

- IS

- MPA

- MB

- MH

minutes

milliVolts

5.77.79.711.713.715.717.719.721.723.725.727.729.731.733.735.737.739.741.743.745.747.749.751.753.755.757.759.761.763.765.767.769.771.773.775.777.779.781.783.785.787.789.791.793.795.797.799.7 -33.0

31.1

95.2

159.3

223.3

Close-up

- MPA

- MB

- MH

minutes

milliVolts

28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.0 52.0 54.0 0.0

4.5

9.1

13.6

18.1

Fig. 1. Chromatogram of a typical Sauvignon blanc wine (MPA = 3-mercaptopropyl acetate; MB = 3-mercaptobutanol; MH = 3-mercaptohexanol).


Progress report 45

- IS

- ME

- MPA

- MB

- MH

minutes

milliVolts

0.02.04.06.08.010.012.014.016.018.020.022.024.026.028.030.032.034.036.038.040.042.044.046.048.050.052.054.056.058.060.062.064.066.068.070.072.074.076.078.080.082.084.086.088.090.092.094.096.098.0-150.0

280.0

710.0

1140.0

1570.0

2000.0

Close-up

- ME

- MPA

- MB

- MH

minutes

milliVolts

28.4 30.4 32.4 34.4 36.4 38.4 40.4 42.4 44.4 46.4 48.4 50.4 52.4 14.2

87.9

161.6

235.4

309.1

382.9

Fig. 2. Chromatogram of a typical Cabernet Sauvignon wine (ME = 2-mercaptoethanol; MPA = 3-mercaptopropyl acetate; MB = 3-mercaptobutanol; MH = 3-mercaptohexanol).


Progress report 46

- ME

- MPA

- MB

- MH

minutes

milliVolts

3.65.67.69.611.613.615.617.619.621.623.625.627.629.631.633.635.637.639.641.643.645.647.649.651.653.655.657.659.661.663.665.667.669.671.673.675.677.679.681.683.685.687.689.691.693.695.697.699.6 -20.6

248.0

516.6

785.2

1053.8

1322.3

Close-up

- ME

- MPA

- MB - MH

minutes

milliVolts

28.3 30.3 32.3 34.3 36.3 38.3 40.3 42.3 44.3 46.3 48.3 50.3 52.3 25.3

106.0

186.7

267.4

348.1

428.8

Fig. 3. Chromatogram of a typical Merlot wine (ME = 2-mercaptoethanol; MPA = 3-mercaptopropyl acetate; MB = 3-mercaptobutanol; MH = 3-mercaptohexanol).


Progress report 47

ADDENDUM 4

Aroma profiles for each cultivar and each method


Progress report 48




Progress report 49

Table 1. Aroma profile of South African Sauvignon blanc wines (2005, 2006 and 2007 vintages; all regions; 203 wines).

Aroma Compounds Mean Minimum Maximum

1 (isobutyl acetate) 0.16 0.00 0.69

2 0.03 0.00 0.24

3 0.03 0.00 0.41

4 (isobutanol) 0.20 0.06 1.06

5 (isoamyl acetate) 6.45 1.80 18.22

6 (isoamyl alcohol) 4.05 1.80 13.50

7 (ethyl hexanoate) 5.61 3.13 14.34

8 (hexyl acetate) 1.40 0.29 4.10

9 0.03 0.00 0.18

10 0.02 0.00 0.09

11 0.01 0.00 0.11

12 0.02 0.00 0.51

13 (1-hexanol) 0.14 0.00 0.71

14 0.04 0.01 0.09

15 (ethyl octanoate) 71.17 43.55 157.56

16 0.19 0.09 1.31

17 0.03 0.00 0.15

18 0.02 0.00 0.45

19 0.07 0.00 0.29

20 0.08 0.01 0.43

21 0.09 0.02 0.73

22 0.03 0.00 1.16

23 (ethyl decanoate) 100.00 100.00 100.00

24 0.91 0.02 4.14

25 (diethyl succinate) 0.04 0.00 0.54

26 0.37 0.00 11.80

27 0.06 0.00 0.17

28 0.05 0.00 0.36

29 0.03 0.00 0.18

30 0.02 0.00 0.17

31 (2-phenylethyl acetate) 0.29 0.02 1.21

32 0.05 0.00 0.39

33 5.48 0.26 24.84

34 0.12 0.00 0.55

35 0.41 0.09 3.65

36 (2-phenylethyl alcohol) 0.25 0.00 1.25

37 0.14 0.00 0.86

38 0.02 0.00 0.35

39 0.03 0.00 0.59

40 0.02 0.00 0.28

41 0.02 0.00 0.26

42 0.01 0.00 0.12

43 0.06 0.00 0.22

44 0.17 0.00 3.16

� Peaks : 2, 3, 9 – 12, 14, 16 – 22, 24, 26 – 30, 32 – 35, 37 – 44 = Unknown � All peaks normalised with respect to ethyl decanoate as 100%


Progress report 50

Table 2. Aroma profile of South African Chardonnay wines (2005, 2006 and 2007 vintages; all regions; 134 wines).



2 (isobutanol) 0.16 0.00 0.86




6 (hexyl acetate) 1.02 0.01 3.53

7 (1-hexanol) 0.10 0.02 0.48

8 0.04 0.02 0.29

9 0.02 0.00 0.27

10 (ethyl octanoate) 60.38 30.01 91.80

11 0.13 0.06 0.75

12 0.01 0.00 0.10

13 0.07 0.00 0.28

14 0.05 0.00 0.56

15 0.14 0.00 8.37

16 (ethyl decanoate) 100.00 100.00 100.00

17 0.72 0.00 2.14


19 0.25 0.00 3.17

20 0.08 0.00 0.35

21 0.04 0.00 0.10

22 0.03 0.00 0.20


24 0.04 0.00 0.56

25 7.66 0.43 20.06

26 0.41 0.00 1.33

27 0.03 0.00 0.13


29 0.16 0.00 1.03

30 0.01 0.00 0.15

31 0.02 0.00 0.11

32 0.01 0.00 0.06

33 0.01 0.00 0.07

34 0.00 0.00 0.05

35 0.04 0.00 0.52

36 0.23 0.00 1.34

37 0.03 0.00 1.12

� Peaks : 8, 9, 11 – 15, 17, 19 – 22, 24 – 27, 29 – 37 = Unknown � All peaks normalised with respect to ethyl decanoate as 100%


Progress report 51

Table 3. Aroma profile of South African Pinotage wines (2005, 2006 and 2007 vintages; all regions; 130 wines).



2 (isobutanol) 1.81 0.00 8.71

3 (isoamyl acetate) 14.04 0.00 40.53

4 0.18 0.00 7.42

5 0.02 0.00 0.90

6 0.11 0.00 2.64

7 0.02 0.00 1.15

8 (isoamyl alcohol) 21.54 1.97 68.71


10 (hexyl acetate) 0.64 0.00 4.05

11 0.01 0.00 0.53

12 0.53 0.00 2.55

13 (1-hexanol) 0.50 0.06 2.00

14 0.11 0.00 0.24

15 (ethyl octanoate) 74.87 43.47 119.36

16 0.36 0.00 6.26

17 0.22 0.00 0.79

18 (ethyl decanoate) 100.00 100.00 100.00

19 1.35 0.50 2.37

20 0.16 0.00 1.52

21 1.63 0.02 5.66

22 3.49 0.03 15.28

23 0.14 0.00 0.41


25 0.13 0.00 0.55

26 10.09 2.65 27.40

27 0.67 0.06 1.69

28 0.08 0.00 0.77


30 1.51 0.00 7.53

31 0.56 0.00 2.51

32 0.31 0.00 1.75

33 0.89 0.12 2.74

34 2.29 0.00 15.77

35 1.04 0.00 33.99

� Peaks : 4 – 7, 11 – 12, 14, 16 – 17, 19 – 23, 25 – 28, 30 – 35 = Unknown � All peaks normalised with respect to ethyl decanoate as 100%


Progress report 52

Table 4. Aroma profile of South African Shiraz wines (2005, 2006 and 2007 vintages; all regions; 165 wines).



2 0.21 0.00 2.29

3 0.18 0.00 1.45

4 (isobutanol) 3.80 0.00 20.47

5 (isoamyl acetate) 11.43 2.65 34.57

6 (isoamyl alcohol) 54.48 12.70 164.32


8 (hexyl acetate) 0.59 0.00 1.60

9 0.30 0.00 5.59

10 0.81 0.00 4.10

11 (1-hexanol) 1.14 0.00 5.62

12 0.17 0.00 0.77

13 (ethyl octanoate) 77.92 39.11 174.07

14 0.58 0.15 2.09

15 0.34 0.00 4.20

16 (ethyl decanoate) 100.00 100.00 100.00

17 1.84 0.00 5.40

18 0.27 0.00 1.80

19 3.61 0.46 14.36

20 0.14 0.00 5.13

21 2.02 0.00 11.86

22 0.11 0.00 0.37

23 0.17 0.00 0.73

24 0.17 0.00 1.18

25 0.10 0.00 0.91

26 0.45 0.00 2.56

27 7.14 0.00 25.05

28 3.77 0.00 22.19

29 0.38 0.00 2.04

30 1.68 0.00 20.06


32 2.06 0.00 21.85

33 0.35 0.00 1.84

34 0.75 0.00 2.64

35 0.18 0.00 1.52

36 0.11 0.00 0.69

37 1.85 0.00 8.96

38 1.04 0.00 10.18

� Peaks : 2 – 3, 9 – 10, 12, 14 – 15, 17 – 30, 32 – 38 = Unknown � All peaks normalised with respect to ethyl decanoate as 100%


Progress report 53

Table 5. Aroma profile of South African Cabernet Sauvignon wines (2005, 2006 and 2007 vintages; all regions; 172 wines).

Aroma Compounds Mean Minimum Maximum 1 (isobutyl acetate) 0.77 0.00 6.58

2 0.27 0.00 2.06

3 0.25 0.00 1.84

4 (isobutanol) 3.79 0.04 11.06

5 (isoamyl acetate) 10.27 2.30 27.89

7 0.05 0.00 5.02

8 0.25 0.00 9.82

10 (isoamyl alcohol) 65.12 17.30 187.44

11 (ethyl hexanoate) 9.00 3.90 21.24

12 (hexyl acetate) 0.38 0.00 1.91

13 0.15 0.00 0.66

14 0.68 0.00 2.91

15 (1-hexanol) 1.22 0.34 5.51

16 0.15 0.00 0.43

17 (ethyl octanoate) 77.70 37.08 122.36

18 0.63 0.16 5.25

19 0.05 0.00 0.42

20 0.11 0.00 0.82

21 0.32 0.00 1.84

22 0.18 0.00 1.19

23 0.27 0.00 0.88 24 (ethyl decanoate) 100.00 100.00 100.00

25 2.68 0.55 4.92

26 0.12 0.00 1.65

27 3.68 0.45 10.33

28 0.05 0.00 1.00

29 2.49 0.00 25.37

30 0.12 0.00 0.48

31 0.16 0.00 0.67

32 0.19 0.00 0.85

33 0.11 0.00 1.08

34 0.59 0.00 3.13

35 0.13 0.00 1.41

36 0.19 0.00 0.96

37 9.28 0.00 25.32

38 1.11 0.00 2.67


40 10.42 1.26 35.91

43 0.07 0.00 0.78

44 0.04 0.00 0.61

45 0.09 0.00 0.44

47 0.47 0.00 1.84

48 0.80 0.00 3.18

49 0.11 0.00 0.57

50 1.75 0.00 9.09

51 0.12 0.00 4.17

52 0.43 0.00 6.40

53 0.35 0.00 2.81

� Peaks : 2 – 3, 7 – 8, 13 – 14, 16, 18 – 23, 25 – 38, 40 – 53 = Unknown � All peaks normalised with respect to ethyl decanoate as 100%


Progress report 54

Table 6. Aroma profile of South African Merlot wines (2005, 2006 and 2007 vintages; all regions; 103 wines).



2 0.15 0.00 0.60

3 0.17 0.00 0.89

4 (isobutanol) 2.81 0.47 12.65


6 (isoamyl alcohol) 46.48 10.00 198.84


8 (hexyl acetate) 0.23 0.00 0.99

9 0.12 0.03 0.46

10 0.47 0.00 2.03

11 (1-hexanol) 0.69 0.16 2.87

12 0.16 0.08 0.38

13 0.20 0.00 7.73

14 (ethyl octanoate) 73.47 46.17 148.55

15 0.52 0.22 1.25

16 0.13 0.00 0.33

17 (ethyl decanoate) 100.00 100.00 100.00

18 2.41 1.13 4.86

19 0.27 0.00 1.50

20 2.52 0.32 9.45

21 2.36 0.09 14.37

22 0.16 0.06 0.46

23 0.13 0.00 0.64

24 0.41 0.09 3.17

25 0.11 0.00 0.44

26 0.04 0.00 0.26

27 0.16 0.00 2.20

28 10.88 0.12 23.42

29 1.19 0.16 6.20

30 0.16 0.00 1.32

31 7.33 0.92 35.15

32 0.79 0.00 6.47

33 0.11 0.00 1.50

34 0.10 0.00 0.72

35 0.08 0.00 0.97

36 0.05 0.00 0.31

37 0.07 0.00 0.69

38 0.48 0.00 2.98

39 0.79 0.10 3.20

� Peaks : 2 – 3, 9 – 10, 12 – 13, 15 – 16, 18 – 39 = Unknown � All peaks normalised with respect to ethyl decanoate as 100%


Progress report 55




Progress report 56



Methoxypyrazines (ng/L) Mean Minimum Maximum

2-Isobutyl-3-methoxypyrazine (IBMP) 11.98 2.12 63.36

2-Isopropyl-3-methoxypyrazine (IPMP) 0.09 0.00 2.83

2-sec-Butyl-3-methoxypyrazine (SBMP) 0.40 0.00 7.57











Progress report 57




Progress report 58

Table 1. Aroma profile of South African Sauvignon blanc wines (2005, 2006 and 2007 vintages; all regions; 181 wines). Aroma compounds (mg/L) Mean Minimum Maximum

Unknown 1 0.12 0.00 1.89

Unknown 2 0.02 0.00 0.28

Unknown 3 1.54 0.00 16.09

Unknown 4 1.28 0.00 5.57

Unknown 5 0.44 0.00 1.75

Unknown 6 0.02 0.00 0.13

Unknown 7 0.02 0.00 0.50

Unknown 8 0.05 0.00 0.37

Unknown 9 1.23 0.00 7.09

Unknown 10 4.98 0.00 20.32

Unknown 11 4.15 0.00 13.11

Unknown 13 2.19 0.00 9.90

Unknown 14 0.56 0.00 4.91

Unknown 15 1.04 0.00 6.84

Unknown 16 0.41 0.00 4.75

2-Phenylethanol 13.15 6.89 32.78

2-Phenylethyl Acetate 0.24 0.00 1.47

Acetic Acid 419.84 80.91 1191.02

Butanol 0.76 0.00 2.54

Butyric Acid 1.76 0.78 3.81

Decanoic Acid 1.74 0.43 5.79

Diethyl Succinate 0.43 0.00 4.89

Ethyl Hexanoate 39.03 0.34 223.58

Ethyl Acetate 49.68 0.20 145.08

Ethyl Butyrate 0.27 0.00 0.80

Ethyl Caprate 0.51 0.00 2.58

Ethyl Caprylate 0.71 0.27 1.77

Ethyl Lactate 9.53 0.00 29.52

Hexanoic Acid 5.67 2.85 13.70

Hexanol 1.25 0.13 4.11

Hexyl Acetate 0.55 0.00 2.48

Isoamyl Acetate 5.63 1.09 18.45

Isoamyl Alcohol 176.65 115.40 394.35

Isobutanol 18.58 2.26 66.32

Isobutyric Acid 0.98 0.00 2.74

Isovaleric Acid 0.82 0.15 2.25

Methanol 67.74 16.02 180.47

Octanoic Acid 6.74 1.73 12.24

Propanol 33.88 10.34 82.65

Propionic Acid 6.57 1.12 43.01

Valeric Acid 0.01 0.00 0.37


Progress report 59

Table 2. Aroma profile of South African Chardonnay wines (2005, 2006 and 2007 vintages; all regions; 120 wines). Aroma compounds (mg/L) Mean Minimum Maximum

Unknown 1 0.02 0.00 1.64

Unknown 2 0.04 0.00 0.15

Unknown 3 3.17 0.00 111.09

Unknown 4 1.26 0.00 6.71

Unknown 5 0.64 0.00 4.02

Unknown 6 0.01 0.00 0.21

Unknown 7 0.06 0.00 0.83

Unknown 8 0.11 0.00 0.64

Unknown 9 1.69 0.00 6.26

Unknown 10 6.74 0.00 23.18

Unknown 11 4.48 0.00 14.48

Unknown 13 1.23 0.00 6.66

Unknown 14 0.37 0.00 1.54

Unknown 15 1.01 0.00 7.10

Unknown 16 0.38 0.00 2.04



Acetic Acid 337.59 4.09 894.12

Butanol 0.82 0.00 2.13





Ethyl Acetate 67.45 0.00 185.78






Hexanol 1.00 0.02 2.73



Isoamyl Alcohol 151.42 1.39 394.93

Isobutanol 15.78 0.65 45.03



Methanol 82.98 0.69 482.00


Propanol 50.61 0.75 149.27




Progress report 60

Table 3. Aroma profile of South African Pinotage wines (2005, 2006 and 2007 vintages; all regions; 121 wines). Aroma compounds (mg/L) Mean Minimum Maximum

Unknown 1 0.11 0.00 1.19

Unknown 2 0.05 0.00 0.18

Unknown 3 16.76 0.00 201.64

Unknown 4 1.70 0.00 7.91

Unknown 5 1.67 0.00 8.61

Unknown 6 0.02 0.00 0.32

Unknown 7 0.05 0.00 0.39

Unknown 8 0.30 0.00 2.74

Unknown 9 1.89 0.00 12.03

Unknown 10 9.37 0.00 32.83

Unknown 11 1.56 0.00 3.85

Unknown 13 0.41 0.00 1.73

Unknown 14 0.52 0.04 9.65

Unknown 15 6.18 0.00 49.91

Unknown 16 0.97 0.00 4.36



Acetic Acid 587.97 246.69 1280.05

Butanol 1.52 0.01 2.80





Ethyl Acetate 62.09 0.00 183.58




Ethyl Lactate 130.10 15.75 402.15


Hexanol 1.09 0.18 2.35



Isoamyl Alcohol 213.08 119.55 331.12

Isobutanol 30.34 3.40 62.00



Methanol 149.46 44.19 284.80


Propanol 93.46 4.81 285.91




Progress report 61

Table 4. Aroma profile of South African Shiraz wines (2005, 2006 and 2007 vintages; all regions; 162 wines). Aroma compounds (mg/L) Mean Minimum Maximum

Unknown 1 0.27 0.00 1.29

Unknown 2 0.09 0.00 0.34

Unknown 3 0.08 0.00 0.52

Unknown 4 1.21 0.00 6.63

Unknown 5 0.99 0.00 5.38

Unknown 6 0.03 0.00 0.26

Unknown 7 0.04 0.00 0.37

Unknown 8 0.53 0.00 3.73

Unknown 9 1.13 0.05 3.93

Unknown 10 13.81 0.00 73.22

Unknown 11 0.88 0.00 3.97

Unknown 13 0.72 0.04 3.39

Unknown 14 0.71 0.00 2.61

Unknown 15 17.71 0.41 91.13

Unknown 16 1.75 0.00 7.68

2-Phenylethanol 42.47 14.31 96.27


Acetic Acid 543.13 225.27 945.58

Butanol 1.81 0.00 3.57





Ethyl Acetate 68.23 20.19 157.07




Ethyl Lactate 101.87 23.54 214.24


Hexanol 1.88 0.36 5.47



Isoamyl Alcohol 310.86 174.34 603.35

Isobutanol 38.06 4.26 92.70



Methanol 223.11 0.00 439.06


Propanol 48.43 2.62 178.40




Progress report 62

Table 5. Aroma profile of South African Cabernet Sauvignon wines (2005, 2006 and 2007 vintages; all regions; 161 wines). Aroma compounds (mg/L) Mean Minimum Maximum

Unknown 1 0.18 0.00 1.75

Unknown 2 0.15 0.00 0.60

Unknown 3 0.09 0.00 0.52

Unknown 4 0.25 0.00 9.17

Unknown 5 0.42 0.00 1.79

Unknown 6 0.04 0.00 0.45

Unknown 7 0.03 0.00 0.25

Unknown 8 0.94 0.00 3.91

Unknown 9 1.69 0.11 2.63

Unknown 10 37.51 0.00 132.03

Unknown 11 1.09 0.00 3.23

Unknown 13 0.61 0.00 2.38

Unknown 14 0.60 0.00 2.27

Unknown 15 23.15 0.47 104.86

Unknown 16 1.41 0.01 5.96

2-Phenylethanol 68.37 24.47 142.14


Acetic Acid 511.78 230.04 882.61

Butanol 1.90 0.91 5.00





Ethyl Acetate 64.85 3.73 125.67




Ethyl Lactate 102.36 19.64 194.70


Hexanol 1.73 0.68 5.36



Isoamyl Alcohol 380.90 159.49 625.79

Isobutanol 37.69 2.34 115.89



Methanol 193.45 79.80 407.95


Propanol 44.85 3.29 155.10




Progress report 63

Table 6. Aroma profile of South African Merlot wines (2005, 2006 and 2007 vintages; all regions; 158 wines). Aroma compounds (mg/L) Mean Minimum Maximum

Unknown 1 0.21 0.00 1.39

Unknown 2 0.10 0.00 0.41

Unknown 3 0.08 0.00 0.58

Unknown 4 0.87 0.00 5.46

Unknown 5 0.97 0.00 7.49

Unknown 6 0.03 0.00 0.33

Unknown 7 0.02 0.00 0.40

Unknown 8 0.82 0.00 4.25

Unknown 9 1.74 0.00 2.96

Unknown 10 34.93 0.00 114.62

Unknown 11 0.81 0.00 2.82

Unknown 13 0.83 0.00 8.76

Unknown 14 1.51 0.00 11.56

Unknown 15 20.46 0.57 90.01

Unknown 16 1.60 0.02 6.80

2-Phenylethanol 60.42 10.14 155.41


Acetic Acid 483.95 39.84 1324.16

Butanol 1.83 1.04 3.53





Ethyl Acetate 42.58 0.00 145.54




Ethyl Lactate 89.51 4.13 194.67


Hexanol 1.23 0.26 4.39



Isoamyl Alcohol 344.96 174.43 642.33

Isobutanol 59.42 24.88 124.30



Methanol 238.56 89.21 406.66


Propanol 40.27 9.09 150.41




Progress report 64




Progress report 65


Mercaptans Mean Minimum Maximum

2-Mercaptoethanol (ME)(µg/L) 7.63 0.00 141.47

3-Mercaptopropyl acetate (MPA)(ng/L) 282.78 0.00 7641.82

3-Mercapto-3-methylbutan-1-ol (MB)(ng/L) 281.16 0.00 7733.86

3-Mercaptohexan-1-ol (MH)(ng/L) 469.24 0.00 6837.73














Progress report 66

ADDENDUM 5

Aroma profiles for each cultivar, from two regions and each method


Progress report 67




Progress report 68

Table 1. Aroma profiles of South African Sauvignon blanc wines (2005, 2006 and 2007 vintages; Stellenbosch region = 33 wines and Robertson region = 35 wines).

Stellenbosch region Aroma Compounds Mean Minimum Maximum


2 0.02 0.00 0.09

3 0.02 0.01 0.08

4 (isobutanol) 0.20 0.08 0.70




8 (hexyl acetate) 1.29 0.3 2.37

9 0.02 0.01 0.06

10 0.02 0.00 0.04

11 0.01 0.00 0.03

12 0.02 0.00 0.09

13 (1-hexanol) 0.11 0.06 0.23

14 0.05 0.03 0.09

15 (ethyl octanoate) 70.6 45.94 102.69

16 0.20 0.09 1.31

17 0.04 0.00 0.11

18 0.02 0.00 0.20

19 0.08 0.03 0.28

20 0.07 0.01 0.13

21 0.09 0.02 0.37

22 0.05 0.00 1.16

23 (ethyl decanoate) 100.00 100.00 100.00

24 0.87 0.53 1.46


26 0.11 0.00 0.58

27 0.06 0.04 0.11

28 0.05 0.02 0.07

29 0.03 0.01 0.05

30 0.03 0.01 0.05


32 0.06 0.01 0.20

33 4.56 0.29 12.53

34 0.14 0.00 0.49

35 0.37 0.23 0.54


37 0.09 0.00 0.62

38 0.03 0.00 0.14

39 0.02 0.00 0.15

40 0.02 0.00 0.12

41 0.02 0.00 0.11

42 0.01 0.00 0.08

43 0.07 0.03 0.16

44 0.12 0.00 0.35


Progress report 69

Table 1 continued Robertson region



2 0.03 0.00 0.24

3 0.02 0.01 0.12

4 (isobutanol) 0.21 0.08 0.82




8 (hexyl acetate) 1.48 0.69 3.88

9 0.03 0.00 0.18

10 0.03 0.01 0.07

11 0.02 0.00 0.05

12 0.03 0.00 0.40

13 (1-hexanol) 0.16 0.06 0.40

14 0.04 0.02 0.09

15 (ethyl octanoate) 70.12 48.18 131.76

16 0.20 0.10 0.62

17 0.03 0.00 0.15

18 0.01 0.00 0.03

19 0.06 0.04 0.13

20 0.07 0.02 0.11

21 0.10 0.02 0.73

22 0.04 0.00 0.56

23 (ethyl decanoate) 100.00 100.00 100.00

24 0.97 0.68 1.48


26 0.13 0.01 0.90

27 0.05 0.00 0.15

28 0.05 0.02 0.11

29 0.03 0.00 0.08

30 0.02 0.00 0.06


32 0.04 0.01 0.24

33 5.85 0.47 13.43

34 0.09 0.00 0.55

35 0.42 0.29 0.70


37 0.20 0.04 0.61

38 0.02 0.00 0.21

39 0.04 0.00 0.38

40 0.02 0.00 0.13

41 0.01 0.00 0.03

42 0.01 0.00 0.12

43 0.06 0.03 0.19

44 0.17 0.01 1.09

� Peaks : 2, 3, 9 – 12, 14, 16 – 22, 24, 26 – 30, 32 – 35, 37 – 44 = Unknown � All peaks normalised with respect to ethyl decanoate as 100%


Progress report 70

Table 2. Aroma profiles of South African Chardonnay wines (2005, 2006 and 2007 vintages; Stellenbosch region = 2 wines and Robertson region = 35 wines). Stellenbosch region



2 (isobutanol) 0.12 0.05 0.18




6 (hexyl acetate) 1.03 0.83 1.23

7 (1-hexanol) 0.09 0.07 0.11

8 0.03 0.03 0.03

9 0.00 0.00 0.01

10 (ethyl octanoate) 58.90 54.16 63.65

11 0.11 0.10 0.13

12 0.02 0.01 0.02

13 0.05 0.04 0.05

14 0.04 0.00 0.08

15 0.04 0.00 0.08

16 (ethyl decanoate) 100.00 100.00 100.00

17 0.66 0.57 0.75


19 1.55 0.02 3.07

20 0.05 0.03 0.07

21 0.06 0.05 0.06

22 0.03 0.03 0.03


24 0.02 0.02 0.02

25 8.97 7.23 10.71

26 0.42 0.36 0.48

27 0.05 0.00 0.09


29 0.10 0.02 0.19

30 0.01 0.00 0.02

31 0.02 0.02 0.02

32 0.01 0.00 0.01

33 0.01 0.00 0.01

34 0.00 0.00 0.00

35 0.06 0.05 0.07

36 0.17 0.16 0.18

37 0.03 0.02 0.04


Progress report 71




2 (isobutanol) 0.16 0.06 0.54




6 (hexyl acetate) 0.88 0.01 1.77

7 (1-hexanol) 0.10 0.03 0.45

8 0.04 0.02 0.07

9 0.01 0.00 0.09

10 (ethyl octanoate) 60.95 47.80 91.80

11 0.13 0.08 0.21

12 0.01 0.00 0.07

13 0.07 0.01 0.20

14 0.04 0.00 0.14

15 0.06 0.00 0.16

16 (ethyl decanoate) 100.00 100.00 100.00

17 0.69 0.47 0.99


19 0.14 0.00 0.71

20 0.08 0.00 0.25

21 0.04 0.00 0.06

22 0.02 0.00 0.08


24 0.04 0.00 0.21

25 7.28 2.60 12.43

26 0.37 0.00 1.33

27 0.02 0.00 0.09


29 0.18 0.00 0.78

30 0.01 0.00 0.09

31 0.02 0.00 0.07

32 0.01 0.00 0.03

33 0.00 0.00 0.07

34 0.00 0.00 0.03

35 0.04 0.00 0.12

36 0.23 0.00 1.20

37 0.02 0.00 0.05

� Peaks : 8, 9, 11 – 15, 17, 19 – 22, 24 – 27, 29 – 37 = Unknown � All peaks normalised with respect to ethyl decanoate as 100%


Progress report 72

Table 3. Aroma profiles of South African Pinotage wines (2005, 2006 and 2007 vintages; Stellenbosch region = 13 wines and Robertson region = 11 wines). Stellenbosch region



2 (isobutanol) 1.99 0.62 6.18

3 (isoamyl acetate) 16.38 4.72 31.59

4 0.07 0.00 0.90

5 0.07 0.00 0.90

6 0.29 0.00 2.64

7 0.09 0.00 1.15

8 (isoamyl alcohol) 23.03 1.97 60.89


10 (hexyl acetate) 0.79 0.16 1.90

11 0.03 0.00 0.24

12 0.55 0.00 1.60

13 (1-hexanol) 0.50 0.06 1.59

14 0.10 0.03 0.24

15 (ethyl octanoate) 67.29 49.14 104.67

16 0.25 0.00 0.82

17 0.22 0.05 0.53

18 (ethyl decanoate) 100.00 100.00 100.00

19 1.18 0.50 1.98

20 0.17 0.00 0.48

21 1.91 0.02 5.66

22 4.17 0.06 14.46

23 0.13 0.02 0.34


25 0.19 0.03 0.44

26 9.05 4.11 12.98

27 0.64 0.17 1.28

28 0.08 0.00 0.45


30 1.69 0.00 4.55

31 0.73 0.05 2.51

32 0.43 0.00 0.77

33 0.98 0.12 2.10

34 2.53 0.27 7.90

35 0.92 0.06 5.19


Progress report 73




2 (isobutanol) 2.43 0.31 8.71

3 (isoamyl acetate) 13.56 4.79 28.07

4 0.07 0.00 0.54

5 0.05 0.00 0.43

6 0.13 0.00 0.99

7 0.03 0.00 0.29

8 (isoamyl alcohol) 27.30 5.31 68.71


10 (hexyl acetate) 0.55 0.11 1.19

11 0.05 0.00 0.53

12 0.65 0.07 2.31

13 (1-hexanol) 0.69 0.20 2.00

14 0.13 0.07 0.22

15 (ethyl octanoate) 78.42 52.79 119.36

16 0.39 0.19 0.63

17 0.22 0.07 0.56

18 (ethyl decanoate) 100.00 100.00 100.00

19 1.54 1.03 2.37

20 0.26 0.00 1.17

21 2.03 0.34 4.46

22 3.72 0.34 15.28

23 0.17 0.06 0.33


25 0.09 0.00 0.23

26 12.55 4.70 21.80

27 0.73 0.06 1.38

28 0.20 0.00 0.77


30 2.12 0.00 7.42

31 0.76 0.00 1.52

32 0.30 0.02 0.89

33 1.24 0.28 2.15

34 2.80 0.39 4.35

35 0.72 0.00 1.96

� Peaks : 4 – 7, 11 – 12, 14, 16 – 17, 19 – 23, 25 – 28, 30 – 35 = Unknown � All peaks normalised with respect to ethyl decanoate as 100%


Progress report 74

Table 4. Aroma profiles of South African Shiraz wines (2005, 2006 and 2007 vintages; Stellenbosch region = 16 wines and Robertson region = 24 wines). Stellenbosch region



2 0.18 0.00 1.73

3 0.18 0.00 1.32

4 (isobutanol) 4.67 0.10 16.18

5 (isoamyl acetate) 11.21 5.13 25.04

6 (isoamyl alcohol) 60.21 30.14 164.32


8 (hexyl acetate) 0.55 0.23 1.01

9 0.16 0.05 0.31

10 0.76 0.00 1.39

11 (1-hexanol) 1.05 0.35 2.27

12 0.16 0.00 0.77

13 (ethyl octanoate) 82.25 56.84 106.67

14 0.58 0.29 1.43

15 0.28 0.00 1.21

16 (ethyl decanoate) 100.00 100.00 100.00

17 2.32 1.25 5.40

18 0.42 0.00 0.83

19 3.89 0.46 11.67

20 0.12 0.00 1.21

21 3.74 0.00 11.86

22 0.13 0.05 0.19

23 0.18 0.00 0.48

24 0.22 0.00 1.18

25 0.08 0.00 0.27

26 0.61 0.00 1.43

27 8.07 0.21 15.60

28 2.63 0.00 12.79

29 0.41 0.00 1.64

30 1.74 0.11 17.04


32 2.26 0.00 17.29

33 0.45 0.00 0.89

34 0.71 0.00 1.83

35 0.17 0.00 1.16

36 0.12 0.00 0.27

37 1.90 0.00 4.60

38 1.44 0.00 10.18


Progress report 75




2 0.23 0.00 1.66

3 0.27 0.00 1.34

4 (isobutanol) 3.95 0.00 9.61

5 (isoamyl acetate) 13.05 2.90 23.17

6 (isoamyl alcohol) 61.40 12.70 132.02


8 (hexyl acetate) 0.69 0.10 1.36

9 0.42 0.05 1.42

10 0.95 0.00 4.10

11 (1-hexanol) 1.33 0.00 3.15

12 0.16 0.00 0.33

13 (ethyl octanoate) 68.62 39.11 103.36

14 0.57 0.19 1.02

15 0.31 0.00 1.37

16 (ethyl decanoate) 100.00 100.00 100.00

17 1.58 0.00 3.32

18 0.26 0.00 1.46

19 3.99 0.97 14.36

20 0.13 0.00 0.59

21 1.16 0.00 4.84

22 0.12 0.00 0.26

23 0.15 0.00 0.66

24 0.16 0.00 0.63

25 0.10 0.00 0.28

26 0.45 0.00 1.73

27 6.57 0.00 15.65

28 5.40 0.00 20.92

29 0.38 0.00 1.19

30 1.67 0.00 6.56


32 3.68 0.00 21.85

33 0.22 0.00 1.03

34 0.77 0.00 2.64

35 0.25 0.00 0.95

36 0.09 0.00 0.53

37 1.59 0.00 8.96

38 1.12 0.00 5.41

� Peaks : 2 – 3, 9 – 10, 12, 14 – 15, 17 – 30, 32 – 38 = Unknown � All peaks normalised with respect to ethyl decanoate as 100%


Progress report 76

Table 5. Aroma profiles of South African Cabernet Sauvignon wines (2005, 2006 and 2007 vintages; Stellenbosch region = 28 wines and Robertson region = 29 wines). Stellenbosch region


2 0.35 0.04 1.81

3 0.37 0.06 1.84

4 (isobutanol) 4.23 1.76 9.55

5 (isoamyl acetate) 11.81 3.58 27.89

7 0.04 0.00 0.32

8 0.20 0.00 2.04

10 (isoamyl alcohol) 78.51 30.59 187.44

11 (ethyl hexanoate) 8.71 4.17 21.24

12 (hexyl acetate) 0.42 0.00 1.80

13 0.15 0.00 0.37

14 0.81 0.19 2.63

15 (1-hexanol) 1.24 0.57 5.51

16 0.13 0.00 0.29

17 (ethyl octanoate) 77.20 46.48 120.05

18 0.56 0.16 1.33

19 0.05 0.00 0.25

20 0.15 0.00 0.82

21 0.38 0.00 1.84

22 0.28 0.00 1.19

23 0.25 0.00 0.44

24 (ethyl decanoate) 100.00 100.00 100.00

25 2.83 0.81 4.26

26 0.04 0.00 0.15

27 4.19 1.46 9.80

28 0.01 0.00 0.11

29 2.79 0.00 8.70

30 0.13 0.00 0.47

31 0.16 0.00 0.63

32 0.19 0.00 0.85

33 0.14 0.00 0.69

34 0.75 0.00 2.21

35 0.09 0.00 0.34

36 0.23 0.00 0.96

37 7.54 0.00 18.77

38 1.03 0.35 1.72


40 14.66 4.86 35.91

43 0.12 0.00 0.56

44 0.05 0.00 0.45

45 0.08 0.00 0.26

47 0.58 0.00 1.17

48 0.62 0.00 1.54

49 0.11 0.00 0.31

50 1.93 0.00 5.34

51 0.11 0.00 0.30

52 0.32 0.00 2.10

53 0.57 0.00 2.79

Progress report 77




2 0.26 0.02 1.50

3 0.23 0.03 0.87

4 (isobutanol) 4.17 1.50 11.06

5 (isoamyl acetate) 11.25 3.43 27.73

7 0.03 0.00 0.50

8 0.21 0.00 3.41

10 (isoamyl alcohol) 70.17 27.45 172.71

11 (ethyl hexanoate) 9.45 4.17 16.66

12 (hexyl acetate) 0.42 0.00 1.16

13 0.19 0.05 0.66

14 0.63 0.00 1.52

15 (1-hexanol) 1.62 0.46 3.70

16 0.16 0.07 0.28

17 (ethyl octanoate) 74.19 43.22 120.02

18 0.65 0.21 1.26

19 0.04 0.00 0.21

20 0.09 0.00 0.40

21 0.30 0.00 1.30

22 0.17 0.00 0.46

23 0.32 0.19 0.88

24 (ethyl decanoate) 100.00 100.00 100.00

25 2.77 1.51 4.29

26 0.15 0.00 0.61

27 3.57 0.45 9.30

28 0.15 0.00 1.00

29 1.44 0.11 4.86

30 0.14 0.04 0.48

31 0.15 0.00 0.30

32 0.20 0.07 0.54

33 0.12 0.00 0.69

34 0.86 0.00 2.63

35 0.14 0.00 0.57

36 0.22 0.00 0.59

37 11.06 6.15 25.32

38 1.30 0.62 2.67


40 10.66 3.33 30.21

43 0.08 0.00 0.54

44 0.06 0.00 0.36

45 0.08 0.00 0.32

47 0.58 0.07 1.78

48 1.02 0.11 3.04

49 0.16 0.00 0.57

50 2.66 0.00 9.09

51 0.24 0.00 4.17

52 0.43 0.00 3.09

53 0.35 0.00 1.28

� Peaks : 2 – 3, 7 – 8, 13 – 14, 16, 18 – 23, 25 – 38, 40 – 53 = Unknown � All peaks normalised with respect to ethyl decanoate as 100%

Progress report 78


Table 6. Aroma profiles of South African Merlot wines (2005, 2006 and 2007 vintages; Stellenbosch region = 14 wines and Robertson region = 13 wines). Stellenbosch region



2 0.19 0.05 0.52

3 0.21 0.08 0.59

4 (isobutanol) 2.43 1.40 4.82


6 (isoamyl alcohol) 43.40 25.07 125.70


8 (hexyl acetate) 0.22 0.04 0.69

9 0.11 0.05 0.34

10 0.47 0.15 2.03

11 (1-hexanol) 0.52 0.18 1.67

12 0.13 0.08 0.19

13 0.13 0.03 1.04

14 (ethyl octanoate) 64.10 46.17 75.42

15 0.41 0.28 0.62

16 0.11 0.06 0.29

17 (ethyl decanoate) 100.00 100.00 100.00

18 2.18 1.57 3.38

19 0.26 0.00 0.45

20 2.37 0.69 9.45

21 2.15 0.54 6.29

22 0.15 0.08 0.31

23 0.12 0.00 0.36

24 0.56 0.13 1.55

25 0.10 0.00 0.29

26 0.05 0.00 0.13

27 0.25 0.00 1.80

28 9.58 5.29 15.47

29 1.07 0.74 1.87

30 0.19 0.00 0.42

31 7.52 2.76 24.61

32 0.64 0.00 2.96

33 0.16 0.00 1.50

34 0.09 0.00 0.45

35 0.11 0.00 0.97

36 0.05 0.00 0.26

37 0.08 0.00 0.69

38 0.69 0.13 2.49

39 0.79 0.17 3.20

Progress report 79





2 0.17 0.05 0.60

3 0.20 0.06 0.89

4 (isobutanol) 3.24 1.34 10.43

5 (isoamyl acetate) 10.48 3.43 40.74

6 (isoamyl alcohol) 57.88 20.01 198.84


8 (hexyl acetate) 0.31 0.05 0.99

9 0.18 0.07 0.46

10 0.43 0.00 1.14

11 (1-hexanol) 0.91 0.41 2.87

12 0.14 0.08 0.21

13 0.08 0.03 0.19

14 (ethyl octanoate) 69.90 48.72 112.15

15 0.62 0.29 1.25

16 0.15 0.00 0.33

17 (ethyl decanoate) 100.00 100.00 100.00

18 2.51 1.33 4.29

19 0.45 0.12 1.50

20 2.55 0.32 4.91

21 2.23 0.16 7.33

22 0.14 0.07 0.23

23 0.17 0.00 0.46

24 0.63 0.18 3.17

25 0.18 0.10 0.44

26 0.06 0.00 0.26

27 0.14 0.00 0.47

28 11.47 4.75 21.08

29 1.24 0.54 2.05

30 0.24 0.00 1.32

31 9.30 3.19 35.15

32 1.05 0.08 4.19

33 0.10 0.00 0.43

34 0.11 0.00 0.48

35 0.06 0.00 0.30

36 0.07 0.00 0.31

37 0.09 0.00 0.26

38 0.59 0.00 2.98

39 0.97 0.19 2.01

� Peaks : 2 – 3, 9 – 10, 12 – 13, 15 – 16, 18 – 39 = Unknown � All peaks normalised with respect to ethyl decanoate as 100%

Progress report 80




Progress report 81


Table 1. Aroma profiles of South African Sauvignon blanc wines (2005, 2006 and 2007 vintages; Stellenbosch region = 37 wines and Robertson region = 40 wines). Stellenbosch region





Robertson region










Robertson region





Progress report 82







Robertson region





Progress report 83




Progress report 84


Table 1. Aroma profiles of South African Sauvignon blanc wines (2005, 2006 and 2007 vintages; Stellenbosch region = 35 wines and Robertson region = 40 wines).

Stellenbosch region

Aroma compounds (mg/L) Mean Minimum Maximum

Unknown 1 0.08 0.00 1.36

Unknown 2 0.02 0.00 0.09

Unknown 3 4.04 0.00 16.09

Unknown 4 1.86 0.00 5.57

Unknown 5 0.42 0.00 1.51

Unknown 6 0.02 0.00 0.13

Unknown 7 0.01 0.00 0.27

Unknown 8 0.10 0.00 0.37

Unknown 9 1.32 0.00 5.96

Unknown 10 6.81 0.00 20.32

Unknown 11 4.42 0.00 11.85

Unknown 13 1.83 0.05 7.49

Unknown 14 0.52 0.00 2.79

Unknown 15 0.87 0.00 4.41

Unknown 16 0.23 0.00 1.73



Acetic Acid 440.57 175.58 863.24

Butanol 0.93 0.00 2.07





Ethyl Acetate 55.40 0.32 135.98






Hexanol 0.94 0.13 2.87



Isoamyl Alcohol 171.88 115.40 371.95

Isobutanol 18.89 2.66 29.54



Methanol 76.51 22.38 166.45


Propanol 37.61 18.54 72.06



Progress report 85


Table 1 continued

Robertson region


Unknown 1 0.18 0.00 1.89

Unknown 2 0.02 0.00 0.11

Unknown 3 1.32 0.00 10.90

Unknown 4 1.21 0.00 3.98

Unknown 5 0.51 0.00 1.75

Unknown 6 0.02 0.00 0.13

Unknown 7 0.01 0.00 0.11

Unknown 8 0.04 0.00 0.32

Unknown 9 1.45 0.00 7.09

Unknown 10 5.60 0.00 15.25

Unknown 11 4.63 0.00 12.46

Unknown 13 2.53 0.00 9.90

Unknown 14 0.64 0.00 4.91

Unknown 15 0.62 0.00 2.18

Unknown 16 0.35 0.00 1.66



Acetic Acid 436.53 139.80 899.19

Butanol 0.75 0.00 1.94





Ethyl Acetate 40.52 0.31 123.42






Hexanol 1.46 0.51 3.59



Isoamyl Alcohol 191.04 124.79 394.35

Isobutanol 16.50 2.26 37.96



Methanol 62.95 16.02 129.44


Propanol 30.68 17.23 64.27



Progress report 86


Table 2. Aroma profiles of South African Chardonnay wines (2005, 2006 and 2007 vintages; Stellenbosch region = 2 wines and Robertson region = 35 wines).

Stellenbosch region


Unknown 1 0.00 0.00 0.00

Unknown 2 0.01 0.01 0.01

Unknown 3 11.00 11.00 11.00

Unknown 4 2.13 2.13 2.13

Unknown 5 0.00 0.00 0.00

Unknown 6 0.00 0.00 0.00

Unknown 7 0.00 0.00 0.00

Unknown 8 0.05 0.05 0.05

Unknown 9 2.52 2.52 2.52

Unknown 10 9.50 9.50 9.50

Unknown 11 7.64 7.64 7.64

Unknown 13 1.14 1.14 1.14

Unknown 14 0.41 0.41 0.41

Unknown 15 0.30 0.30 0.30

Unknown 16 0.54 0.54 0.54



Acetic Acid 391.71 379.93 403.50

Butanol 0.78 0.46 1.10





Ethyl Acetate 70.92 65.19 76.65






Hexanol 0.95 0.71 1.20



Isoamyl Alcohol 138.72 126.20 151.24

Isobutanol 14.12 3.39 24.84



Methanol 57.11 52.52 61.70


Propanol 31.62 30.84 32.41



Progress report 87


Table 2 continued

Robertson region


Unknown 1 0.00 0.00 0.00

Unknown 2 0.05 0.00 0.10

Unknown 3 6.55 0.00 111.09

Unknown 4 1.41 0.00 5.21

Unknown 5 0.68 0.00 1.97

Unknown 6 0.01 0.00 0.21

Unknown 7 0.11 0.00 0.83

Unknown 8 0.13 0.00 0.64

Unknown 9 1.83 0.00 5.92

Unknown 10 6.84 0.00 14.53

Unknown 11 6.40 0.02 14.48

Unknown 13 1.80 0.04 6.38

Unknown 14 0.42 0.00 1.54

Unknown 15 0.68 0.00 7.10

Unknown 16 0.43 0.00 1.50



Acetic Acid 382.56 180.04 894.12

Butanol 0.93 0.00 2.13





Ethyl Acetate 59.68 0.00 185.78






Hexanol 1.07 0.32 2.73



Isoamyl Alcohol 156.67 110.99 292.74

Isobutanol 16.13 2.27 37.96



Methanol 79.36 29.12 156.23


Propanol 54.67 17.89 107.14



Progress report 88


Table 3. Aroma profiles of South African Pinotage wines (2005, 2006 and 2007 vintages; Stellenbosch region = 12 wines and Robertson region = 13 wines).

Stellenbosch region


Unknown 1 0.01 0.00 0.01

Unknown 2 0.05 0.01 0.06

Unknown 3 78.79 0.00 163.97

Unknown 4 1.37 0.00 4.56

Unknown 5 0.92 0.35 1.44

Unknown 6 0.03 0.00 0.11

Unknown 7 0.08 0.04 0.10

Unknown 8 0.81 0.07 1.83

Unknown 9 2.44 0.75 3.20

Unknown 10 9.48 0.01 19.59

Unknown 11 1.82 0.21 3.25

Unknown 13 0.80 0.36 1.31

Unknown 14 0.59 0.25 0.76

Unknown 15 13.14 0.47 49.91

Unknown 16 1.17 0.30 1.59

2-Phenylethanol 20.22 10.99 33.69


Acetic Acid 597.31 307.24 816.88

Butanol 1.75 0.10 2.74





Ethyl Acetate 30.83 0.17 106.09




Ethyl Lactate 134.40 87.51 169.45


Hexanol 1.06 0.42 1.46



Isoamyl Alcohol 219.49 148.38 296.40

Isobutanol 25.04 3.95 62.00



Methanol 153.29 82.35 242.87


Propanol 92.71 39.34 181.55



Progress report 89


Table 3 continued

Robertson region


Unknown 1 0.06 0.00 0.30

Unknown 2 0.06 0.00 0.18

Unknown 3 28.86 0.00 201.64

Unknown 4 2.56 0.04 5.06

Unknown 5 1.25 0.10 3.37

Unknown 6 0.00 0.00 0.01

Unknown 7 0.05 0.00 0.09

Unknown 8 0.30 0.00 1.25

Unknown 9 1.82 0.00 3.41

Unknown 10 13.33 0.15 32.83

Unknown 11 1.75 0.00 3.33

Unknown 13 0.30 0.01 0.60

Unknown 14 0.36 0.15 0.90

Unknown 15 6.17 0.22 37.71

Unknown 16 0.67 0.03 2.19

2-Phenylethanol 24.66 16.62 47.35


Acetic Acid 580.63 447.60 746.82

Butanol 1.47 0.06 2.73





Ethyl Acetate 57.83 0.00 107.91




Ethyl Lactate 124.24 59.18 201.79


Hexanol 1.31 0.27 2.13



Isoamyl Alcohol 248.50 182.05 331.12

Isobutanol 37.08 4.64 57.71



Methanol 170.35 125.80 284.80


Propanol 81.39 50.73 136.36



Progress report 90


Table 4. Aroma profiles of South African Shiraz wines (2005, 2006 and 2007 vintages; Stellenbosch region = 16 wines and Robertson region = 30 wines).

Stellenbosch region


Unknown 1 0.44 0.02 1.25

Unknown 2 0.09 0.03 0.19

Unknown 3 0.07 0.00 0.18

Unknown 4 0.91 0.01 2.53

Unknown 5 1.36 0.00 5.38

Unknown 6 0.05 0.00 0.24

Unknown 7 0.09 0.00 0.22

Unknown 8 1.02 0.00 3.73

Unknown 9 0.69 0.25 2.61

Unknown 10 9.32 0.14 48.87

Unknown 11 0.86 0.13 1.71

Unknown 13 0.70 0.39 1.23

Unknown 14 0.56 0.25 0.88

Unknown 15 37.09 1.89 89.58

Unknown 16 1.72 0.12 3.12

2-Phenylethanol 48.99 23.34 68.20


Acetic Acid 545.35 335.97 704.87

Butanol 2.03 0.05 3.19





Ethyl Acetate 62.46 31.39 107.64




Ethyl Lactate 99.20 44.90 146.64


Hexanol 1.52 0.91 2.17



Isoamyl Alcohol 320.70 255.99 397.80

Isobutanol 29.03 5.43 81.30



Methanol 189.57 103.25 309.39


Propanol 46.96 12.24 105.80



Progress report 91


Table 4 continued

Robertson region


Unknown 1 0.29 0.00 1.29

Unknown 2 0.10 0.01 0.21

Unknown 3 0.05 0.00 0.27

Unknown 4 0.97 0.00 3.96

Unknown 5 0.52 0.00 2.16

Unknown 6 0.05 0.00 0.26

Unknown 7 0.06 0.00 0.37

Unknown 8 0.78 0.00 2.24

Unknown 9 1.09 0.05 2.72

Unknown 10 13.01 0.00 62.91

Unknown 11 0.84 0.08 2.29

Unknown 13 0.53 0.17 1.11

Unknown 14 0.73 0.05 1.96

Unknown 15 25.52 0.51 91.13

Unknown 16 1.58 0.00 4.58

2-Phenylethanol 48.32 20.33 90.43


Acetic Acid 559.49 237.02 889.35

Butanol 1.83 0.14 3.28





Ethyl Acetate 63.93 20.19 85.82




Ethyl Lactate 95.32 27.12 161.02


Hexanol 2.24 0.89 5.47



Isoamyl Alcohol 334.27 229.80 487.64

Isobutanol 39.50 5.18 92.70



Methanol 221.68 112.67 415.66


Propanol 53.70 2.62 138.90



Progress report 92




Unknown 1 0.46 0.03 1.75

Unknown 2 0.12 0.05 0.29

Unknown 3 0.17 0.00 0.42

Unknown 4 0.18 0.00 1.42

Unknown 5 0.76 0.01 1.79

Unknown 6 0.03 0.00 0.15

Unknown 7 0.03 0.00 0.17

Unknown 8 1.17 0.00 3.41

Unknown 9 1.39 0.35 2.49

Unknown 10 29.65 0.14 81.75

Unknown 11 1.04 0.15 2.12

Unknown 13 0.56 0.19 1.64

Unknown 14 0.66 0.15 2.27

Unknown 15 36.44 0.97 104.86

Unknown 16 1.92 0.14 5.96

2-Phenylethanol 83.25 47.70 128.75


Acetic Acid 520.00 230.04 840.43

Butanol 2.28 1.00 4.92





Ethyl Acetate 61.17 32.10 102.70




Ethyl Lactate 104.34 36.89 153.04


Hexanol 1.61 0.75 2.96



Isoamyl Alcohol 414.64 287.69 514.13

Isobutanol 32.53 4.92 76.92



Methanol 188.42 79.80 303.75


Propanol 43.28 10.24 79.29



Progress report 93


Table 5 continued

Robertson region


Unknown 1 0.18 0.02 0.79

Unknown 2 0.14 0.00 0.60

Unknown 3 0.12 0.00 0.52

Unknown 4 0.01 0.00 0.08

Unknown 5 0.31 0.00 1.68

Unknown 6 0.04 0.00 0.33

Unknown 7 0.04 0.00 0.16

Unknown 8 1.31 0.14 3.10

Unknown 9 1.56 0.37 2.54

Unknown 10 35.14 0.00 132.03

Unknown 11 1.00 0.00 3.04

Unknown 13 0.68 0.00 1.57

Unknown 14 0.75 0.05 1.85

Unknown 15 37.46 0.47 85.57

Unknown 16 1.65 0.09 3.92

2-Phenylethanol 71.38 33.41 142.14


Acetic Acid 504.91 278.12 724.86

Butanol 1.91 1.07 3.21





Ethyl Acetate 61.80 43.24 89.34




Ethyl Lactate 96.25 21.14 185.55


Hexanol 1.93 0.87 3.43



Isoamyl Alcohol 392.52 251.16 606.27

Isobutanol 38.73 4.97 101.22



Methanol 213.09 109.55 389.25


Propanol 49.38 17.48 113.31



Progress report 94


Table 6. Aroma profiles of South African Merlot wines (2005, 2006 and 2007 vintages; Stellenbosch region = 20 wines and Robertson region = 27 wines).

Stellenbosch region


Unknown 1 0.34 0.01 1.39

Unknown 2 0.11 0.04 0.33

Unknown 3 0.10 0.00 0.26

Unknown 4 1.17 0.00 5.46

Unknown 5 0.54 0.00 1.57

Unknown 6 0.03 0.00 0.13

Unknown 7 0.02 0.00 0.09

Unknown 8 1.40 0.29 2.52

Unknown 9 2.09 0.45 2.92

Unknown 10 42.02 0.00 78.64

Unknown 11 0.80 0.09 2.10

Unknown 13 0.60 0.18 1.55

Unknown 14 1.11 0.47 3.27

Unknown 15 36.55 1.92 84.80

Unknown 16 1.49 0.17 2.43

2-Phenylethanol 73.43 35.72 120.09


Acetic Acid 465.86 39.84 800.32

Butanol 2.04 1.04 3.37





Ethyl Acetate 30.62 0.00 88.75




Ethyl Lactate 79.81 31.19 194.67


Hexanol 1.09 0.53 4.39



Isoamyl Alcohol 367.44 303.36 446.00

Isobutanol 57.99 41.20 73.25



Methanol 228.10 135.71 406.66


Propanol 36.50 9.63 94.68



Progress report 95


Table 6 continued

Robertson region


Unknown 1 0.31 0.01 1.35

Unknown 2 0.11 0.00 0.41

Unknown 3 0.13 0.00 0.58

Unknown 4 2.00 0.00 4.87

Unknown 5 2.06 0.00 7.49

Unknown 6 0.04 0.00 0.33

Unknown 7 0.05 0.00 0.40

Unknown 8 1.41 0.51 2.37

Unknown 9 1.96 0.30 2.56

Unknown 10 44.88 0.00 114.62

Unknown 11 0.47 0.00 1.55

Unknown 13 0.76 0.00 1.66

Unknown 14 1.03 0.36 4.96

Unknown 15 36.81 0.88 76.77

Unknown 16 1.36 0.82 2.54

2-Phenylethanol 74.16 20.55 155.41


Acetic Acid 404.57 234.57 582.75

Butanol 1.97 1.36 3.39





Ethyl Acetate 29.63 0.00 75.40




Ethyl Lactate 85.91 4.13 149.51


Hexanol 1.33 0.57 2.17



Isoamyl Alcohol 389.01 174.43 607.72

Isobutanol 61.71 32.89 85.42



Methanol 236.12 139.64 318.62


Propanol 42.49 18.60 85.07



Progress report 96




Progress report 97


Table 1. Aroma profiles of South African Sauvignon blanc wines (2005, 2006 and 2007 vintages; Stellenbosch region = 38 wines and Robertson region = 40 wines). Stellenbosch region






Robertson region












Robertson region






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Robertson region






Progress report 99


ADDENDUM 6

New method developed for Wine Industry

Progress report 100



Method: Stir Bar Sorptive Extraction of volatiles in wine

Initially both experimental conditions (instrumental conditions) and the sample preparation (extraction procedure) were thoroughly optimized, validated and applied to real young wine samples supplied by the South African Young Wine Show. Therefore, we report here both the instrumental conditions and sample preparation procedures and we refer them as “Analytical methods 1 and 2”. For the detail of the technique it can be referred to J. Agric. Food Chem. 2007, 55, 8696–8702.

Analytical method 1

Phase I

Experimental Conditions

GC-MS analysis was carried out with an Agilent 6890 GC coupled to a 5973N MS (Agilent Technologies, Palo Alto, CA). A 30 m HP-INNOWax capillary column [(0.250 mm I.D. × 0.5 µm film thickness) (Agilent Technologies)] was used for separating the volatile compounds. The GC oven was held at 30 °C for 2 min, increased to 130 °C at a rate of 4 °C/min and then at 8 °C/min to 250 °C where it was kept for 5 min. Helium was used as the carrier gas with a flow of 1 mL/min in the constant pressure mode. The MS was operated in a scan mode with a scan range of 30–350 amu at 4.45 scans/sec. Spectra were recorded in the electron impact mode (EI) at 70 eV. The MS transfer line, source and quadrupole were at 250, 230, and 150 °C, respectively. Quantitation was performed with total ion chromatograms (TICs) using the sum of all ions for well-separated compounds after careful examination of the peak purity and single ion extraction was applied for closely eluting and minor peaks. Identification was based on comparison of mass spectra with Wiley 275 and NIST 98 libraries as well as retention times of known standards in synthetic wine for all compounds. For comparison with literature data, retention indices (RI) were experimentally determined using a mixture of n-alkanes.

The TDS 2 was carried out with a temperature program from 30°C held for 1 min and raised at 20 °C/min to 260 °C where it was held for 10 min. It was operated in solvent vent mode with a purging time of 3 min and equilibrium time of 1 min. The heated transfer line was set at 300 °C. After desorption, the analytes were cryofocused in a programmed temperature vaporizing inlet (PTV) at -100 °C using liquid nitrogen prior to injection. An empty baffled glass liner was used in the PTV. Solvent vent injection with splitless time of 2 min and purge time of 0.1 min was performed by ramping the PTV from -100 to 270 °C at 12 °C/sec and held for 10 min.

Sample Preparation.

One mL of wine, 100 µL (1.7 mg/L) of 4-methyl-2-pentanol (internal standard), and 1.5 g NaCl was transferred into a 20 mL headspace vial. The volume was made to 6 mL with ultra-pure water of 12% ethanol mixture. The pH was adjusted to 3.2 using a formate buffer. A glass coated magnetic stirrer was added to the mixture. A preconditioned SBSE stir bar Twister (Gerstel, Müllheim a/d Ruhr, Germany) of 10 mm length coated with a 0.5 mm PDMS layer (25 µL) was suspended in the headspace using a glass insert. The vial was sealed with 20 mm aluminum crimp cap and PTFE/silicone molded septa using a hand crimper. The mixture was stirred for 1 hour at room temperature and 1200 rpm. Then the vial was left standing for 3 hours at room temperature. After sampling, the stir bar was removed, dried

Progress report 101


gently with lint free tissue, and placed in a glass tube of 187 mm length, 6 mm o.d. and 4 mm i.d., which then was placed in the TDS-A auto-sampler tray (Gerstel, Müllheim a/d Ruhr, Germany). It was followed by thermal desorption, cryo-trapping, and gas chromatography-mass spectrometry analysis. The stir bars were reconditioned for 30 min at 280 °C under a nitrogen stream flow, and no carry-over was observed. Regularly system blanks were run to confirm cleanliness of the system.

Phase II

In the second phase the analytical method previously reported (mentioned above in phase I) was modified for a better outcome. In this phase both the instrumental conditions and sample preparation procedure from phase I was modified. The time length of the extraction procedure was reduced from 4 hours to 1hour and the sample volume reduced by half. Moreover, tartarate buffer was used instead of formate buffer for pH adjustment to the wines. In a similar way the instrumental conditions were also modified slightly. This method is referred as “Analytical method 2” and is detailed below. For more detailed information of this method it can be referred to J. Agric. Food Chem. 2008, 56, 10225–10236.

Analytical method 2

Instrumental Conditions

The GC-MS analysis was carried out with an Agilent 6890 GC coupled to a 5973N MS (Agilent Technologies). A 30 m HP-INNOWax capillary column [0.250 mm i.d. × 0.5 µm film thickness (Agilent Technologies)] was used for separating the volatile compounds. The GC oven was held at 30 °C for 2 min and increased to 130 °C at a rate of 4 °C/min and then at 8 °C/min to 250 °C, at which it was kept for 5 min. Helium was used as the carrier gas with a flow of 1 mL/min in the constant pressure mode. The MS was operated in a scan mode with a scan range of 30-350 amu at 4.45 scans/s, for peak identification, ion selection, and locating the compounds in the TIC plot. However, for quantitation purposes the MS was operated in the selected ion monitoring (SIM) mode. Three ions with a dwell time of 50 ms for each compound (one quantitative or target ion and two qualitative ions) were selected. Spectra were recorded in the electron impact mode (EI) at 70 eV. The MS transfer line, source, and quadrupole were at 250, 230, and 150 °C, respectively. Identification was based on comparison of mass spectra with Wiley 275 and NIST 98 libraries as well as retention times of known standards in synthetic wine for all compounds. As a complementary identification, linear retention indices (LRI) were experimentally determined using a mixture of n-alkanes and compared with literature values.

The TDS 2 was carried out with a temperature program from 30 °C held for 1 min and raised at 20 °C/min to 260 °C, at which it was kept for 10 min. It was operated in solvent vent mode with a purging time of 3 min and equilibrium time of 1 min. The heated transfer line was set at 300 °C. After desorption, the analytes were cryofocused in a programmed temperature vaporizing injector (PTV) at -100 °C using liquid nitrogen prior to injection. An empty baffled glass liner was used in the PTV. Solvent vent injection with a splitless time of 2 min and a purge time of 0.1 min was performed by ramping the PTV from -100 to 270 at 12 °C/s and held for 10 min.

SBSE Headspace Analysis

A 0.5 mL of wine, 50 mL (1.7 mg/L) of 4-methyl-2-pentanol (internal standard), and 1.5 g of NaCl were transferred to a 20 mL headspace vial. The volume was made up to 6 mL with a blank model wine (a mixture of 12% ethanol in 2 g/L tartarate solution of pH 4.2), which brought the pH of the sample to 3.2. A glass-coated magnetic stirrer was added to the mixture. A preconditioned SBSE stir bar of 10 mm length, coated with a 0.5 mm PDMS layer (25 mL), Twister (Gerstel), was suspended in the headspace using a glass insert, Twister. The vial was sealed with a 20 mm aluminum crimp cap and a PTFE/silicone molded septum using a hand crimper. The mixture was stirred for 1 h at 1200 rpm and controlled room

temperature (23 ± 1 °C). After sampling, the stir bar was removed, dried gently with a lint-free tissue, and placed in a glass tube of 187 mm length, 6 mm o.d., and 4 mm i.d., which then

Progress report 102


was placed in the TDS-A autosampler tray (Gerstel). It was followed by thermal desorption, cryotrapping, and gas chromatography-mass spectrometric analysis. The stir bars were reconditioned for 30 min at 280 °C under a nitrogen stream, and no carry-over was observed. Regular system blanks were run to confirm the cleanliness of the system.

FINALFINAL REPORT REPORT - SAWIS library · Progress report 3 WW0831 – Dr J Marais (ARC...

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