gc x gc voa s african wines

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Application of modern chromatographic technologies for the analysis of volatile compounds in South African wines

by

Berhane Tekle Weldegergis

Dissertation presented for the Degree of

Doctor of Philosophy (Chemistry)at

University of Stellenbosch

Supervisor: Prof. A. M. Crouch Co-supervisor: Dr. A. de Villiers

Stellenbosch December 2009

DECLARATIONBy submitting this dissertation electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the owner of the copyright thereof (unless to the extent explicitly otherwise stated) and that I have not previously in its entirety or in part submitted it for obtaining any qualification.

December 2009

Copyright 2009 Stellenbosch University

All rights reserved

SummaryThe present study was initiated by the wine industry of South Africa to overcome the lack of available information on the flavor and aroma of South African wines. The aim was to develop new analytical methods and improve existing ones for the analysis of volatile compounds in the South African wines. Initially a new analytical method based on stir bar sorptive extraction (SBSE) in the headspace mode for the analysis of 37 pre-selected volatile compounds was developed and validated. Consequently, the method was improved by making important modifications and increasing the number of compounds analyzed to 39. This method was successfully applied to a large number of Pinotage wines of vintages 2005 and 2006. The quantitative data of these wines were subjected to chemometric analysis in order to investigate possible co/variances. A clear distinction was observed between the two vintages, where the 2005 wines were more characterized by wood-related compounds and the 2006 wines by the fermentation compounds. The developed method was further applied to other cultivars of vintage 2005, including two white (Sauvignon Blanc and Chardonnay) and three red (Shiraz, Cabernet Sauvignon and Merlot) cultivars. In a similar fashion, the quantitative data of the six cultivars of vintage of 2005 were analysed by chemometric methods. Significant differences were observed between the two white cultivars and among the four red cultivars. It was shown that among these cultivars, the major role-players were the wood and fermentation related volatiles. A striking observation was the confirmation of the unique character of the Pinotage wines compared to the other red cultivars, mainly influenced by the high level of isoamyl acetate and low level of isoamyl alcohol, the former being categorized as a varietal compound for Pinotage expressed by a fruity (banana) odor. In addition, advanced chromatographic technology in the form of comprehensive twodimensional gas chromatography (GC GC) coupled to time-of-flight mass spectrometry (TOFMS) was investigated for the detailed analysis of volatile compounds in young South African wines. This work focused primarily on Pinotage wines. In the first instance, solid phase micro extraction (SPME) in the headspace mode in combination with GC GC-TOFMS was used. Due to the high resolution and large peak capacity of GC GC, more than 200 compounds previously reported as wine components were identified. These compounds were dominated by the highly

volatile and less polar compounds, mainly due to the characteristics of SPME. In an attempt to further extend these results, another selective extraction method, solid phase extraction (SPE) was used in combination with GC GC-TOFMS analysis. Using this technique, more than 275 compounds, most of them unidentified using the previous method, were detected. These groups of compounds include volatile phenols, lactones as well as mostly aromatic esters and norisoprenoids, which can potentially influence the aroma and flavor of wine. The techniques developed as part of this study have extended our knowledge of the volatile composition of South African wines.

OpsommingHierdie studie is genisieer deur die wyn industrie van Suid-Afrika om die tekort aan beskikbare inligting aangaande wyn aroma van Suid-Afrikaanse wyne te oorkom. Die doel was om nuwe analitiese metodes te ontwikkel en die huidige metodes te verbeter vir die analise van vlugtige verbindings in Suid-Afrikaanse wyne. Oorspronklik is nuwe analitiese metode ontwikkel en gevalideer gebaseer is op stir bar sorptive extraction (SBSE) in die gas fase vir die analise van 37 vooraf geselekteerde vlugtige verbindings. Die metode is verbeter deur belangrike modifikasies aan te bring en die hoeveelheid verbindings wat analiseer word te vermeerder na 39. Hierdie metode is suksesvol aangewend op groot hoeveelheid Pinotage wyne van oesjare 2005 en 2006. Die kwantitatiewe data van hierdie wyne is onderwerp aan verskillende chemometriese analises om moontlike ko-/variasies te ondersoek. Duidelike onderskeid is opgemerk tussen die twee oesjare, waar die 2005 wyne gekarakteriseer is deur hout-verwante verbindings en die 2006 wyne weer meer deur fermentasie verbindings. Die verbeterde metode is verder aangewend vir analiese van ander kultivars van oesjare 2005, wat twee wit (Sauvignon Blanc en Chardonnay) en drie rooies (Shiraz, Cabernet Sauvignon en Merlot) ingesluit het. Die kwantitatiewe data van die ses kultivars van oesjaar 2005 is op soortgelyke wyse geanaliseer deur verskillende chemometriese metodes te gebruik. Beduidende verskille is opgemerk tussen die twee wit kultivars en tussen die vier rooi kultivars. Die hoof rolspelers tussen die ses kultivars was weereens die verbindings wat hout en fermentasie aard het. Die unieke karakter van die Pinotage wyne in vergelyking met die ander rooi kultivars was opvallend. Hierdie wyn word gekarakteriseer deur ho vlakke van isoamiel asetaat en lae vlakke van isoamiel alkohol, waar eersgenoemde gekatogiseer word as verbinding wat vrugte (piesang) geur in Pinotage uitdruk. Verder is gevorderde chromatografiese tegnologie in die vorm van comprehensive two-dimentional gas chromatography (GC x GC) gekoppel met time-of-flight mass spectroscopy (TOFMS) ondersoek vir die analiese van vlugtige verbindings in jong Suid-Afrikaanse wyne. Hierdie werk het hoofsaaklik op Pinotage wyne gefokus. Eerstens is solid phase micro extraction (SPME) in die gas fase gekombineer met GC x GC-TOFMS. As gevolg van die ho resolusie en groot piek kapasiteit van GC x GC is meer as 200 verbindings wat voorheen gerapporteer is as wyn komponente

gedentifiseer. Hierdie verbindings is gedomineer deur ho vlugtige polre verbindings, hoofsaaklik as gevolg van die karaktersitieke van SPME..In poging om die metode verder te verbeter is selektiewe ekstraksie metode naamlik solid phase extraction (SPE) in kombinasie met GC x GC-TOFMS gebruik. Met hierdie tegniek is meer as 275 verbindings gedentifiseer, waarvan die meeste nie met die vorige metode waargeneem is nie. Hierdie verbindings sluit vlugtige fenole, laktone en meestal aromatiese esters en norisoprenodes in, wat moontlik die reuk en smaak van wyn kan benvloed. Die metodes ontwikkel gedurende die studie het nuwe informasie verskaf aangaande die vlugtige komponente teenwoordig in Suid Afrikaanse wyne.

AcknowledgmentsFirst and for most I thank the almighty God for his blessing my life in all my activities. In addition I wish to convey my appreciation to the following people or entities for their diverse contributions throughout the study: All my family for their unconditional love and support. University of Stellenbosch, NRF (THRIP), WineTech South Africa, and Natural Sciences and Engineering Research Council of Canada for financial support. Natural Sciences and Engineering Research Council of Canada for financial support. Prof. Andrew Crouch for his continuous support academically, financially, and socially in the entire study period. Dr. Andre de Villiers for his willingness to be involved as a co-supervisor and for his continuous academic support. All the staff remembers of International Office at Stellenbosch University Ms Linda Uys and Mr Robert Kotz in particular for their all-rounded support. LECO Africa (Dr. Peter Gorst-Allman and Alexander Whaley) for providing instrumentation and software as well as their valuable intellectual contribution. Dr. A.G.J. Tredoux for his continuous support, encouragement academically and socially as well as for his good friendship. Prof. M. Kidd Centre for Statistical Consultation, Stellenbosch University for his competent and enthusiastic assistance with the chemometrical analysis. South African Young Wine Competition for supplying the wine samples. The Institute of Wine Biotechnology, Stellenbosch University, for storing the wine samples. Department of chemistry at University of Pretoria for providing laboratory facilities and, Ms Y Naud in particular for her support. Mr. Tesfamariam Kifle Hagos for his assistance on the data analysis. All my collogues at the Chemistry Department (separations and electrochemistry): Dr. Astrid Buica, Dr. Adriana Stopforth, K.M. Kalili, L.G. Martin, J. Chamier, P. Modiba, and Dr. M.J. Klink for their support. My girlfriend Tanger Thomas for her understanding the weight of the study and supporting me at all levels.

My Son Zerai for making me happy and relaxing after a long and stressful day at work. Daniel, Johannes, Mary, Roger, Deidre, Shafiek and Lucinda for their valuable assistance and friendship.

PrefaceThe study of wine aroma and flavor is quite a complex process, as many of the chemical constituents that are responsible for the sensory property of the wine do not come directly from the grapes; rather their formation is influenced by many other factors. In addition the number and type of compounds already reported in wine are large and certainly one can never cover all in one study. Scientists around the world have already performed numerous studies regarding wine sensory properties, but