Application to real wine samples

After optimizing and validating the method thoroughly, it was applied to the analysis of 64 red wine samples from four different red wine cultivars (Pinotage, Shiraz, Cabernet Sauvignon, and Merlot, 16 samples of each cultivar), and 15 Chardonnay wines, all of vintage 2005. A typical chromatogram of Pinotage wine is presented in Figure 5.1.

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10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

400000

10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

400000

Figure 5.1. A TIC Chromatogram of Pinotage wine vintage 2005 obtained using the optimized method HSSE-TD-GC-MS. Compound identification see Table 5.1.

and Quantitative information see Table 5.2. Concentration of I.S. was 1.7 mg/l. (Conditions see text).

The summary of all the volatile components identified in the wine samples are presented in Table 5.2. Theses compounds mainly belong to esters, alcohols, lower acids, and furans as well as other compounds in lesser amounts belonging to carbonyls, lactones, and phenols. With the current method decanoic acid and 2,6-dimethoxy phenol were unable to identify in all the samples. Moreover, p-cresol was below the LOD in all wines of Shiraz, Cabernet Sauvignon, and Merlot cultivars. The trans-oak-lactone was unidentified in the white wines. Furthermore, it was not detected in all the samples of Cabernet Sauvignon cultivars except in one. Its racemic isomer, cis-oak-lactone, was not determined in all the samples of Cabernet Sauvignon, Merlot, and Chardonnay cultivars.

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84 Table 5.2. Average concentration (mg/l ± SD) of volatile compounds obtained in 79 young South African wine samples of vintage 2005

using the validated method HSSE-TD-GC-MS. (Conditions see text).

Pinotage Shiraz Cabernet Sauvignon Merlot Chardonnay Compounds

Average^a ± SD^b Average^a ± SD^b Average^a ± SD^b Average^a ± SD^b Average^a ± SD^b RI^c Ethyl butyrate 1.04 ± 0.73 0.60 ± 0.17 0.75 ± 0.24 0.79 ± 0.27 2.09 ± 0.59 990 Isobutanol 23.64 ± 11.68 38.87 ± 15.79 36.32 ± 12.74 45.53 ± 29.89 9.61 ± 5.52 1072 Isoamyl acetate 6.27 ± 2.88 4.00 ± 1.55 4.25 ± 1.70 3.65 ± 1.88 10.34 ± 3.53 1098 n-Butanol 3.13 ± 0.80 6.81 ± 3.54 6.22 ± 3.01 5.01 ± 2.34 10.09 ± 5.66 1145 Isoamyl alcohol 183 ± 36.96 207 ± 23.13 268 ± 44.62 264 ± 69.38 159 ± 24.92 1216 Ethyl hexanoate 0.45 ± 0.26 0.30 ± 0.08 0.36 ± 0.10 0.34 ± 0.14 1.14 ± 0.37 1233 Hexyl acetate 0.01± 0.01 0.01 ± 0.01 0.006 ± 0.004 0.003 ± 0.003 0.12 ± 0.05 1278 Acetoin 19.71 ± 10.25 28.51 ± 16.38 19.93 ± 12.12 21.87 ± 11.72 26.54 ± 21.20 1307 Ethyl-D-lactate 230 ± 62.73 184 ± 72.89 220 ± 74.73 208 ± 75.80 51.81 ± 69.80 1364 1-Hexanol 3.55 ± 2.97 4.15 ± 0.98 4.79 ± 1.02 4.06 ± 1.78 6.31 ± 13.49 1372 Ethyl octanoate 0.04 ± 0.03 0.02 ± 0.01 0.024 ± 0.01 0.023 ± 0.01 0.12 ± 0.04 1455 Acetic acid 996 ± 999 1344 ± 846 1395 ± 763 1509 ± 1014 901 ± 499 1476 Furfural 3.73 ± 1.99 7.90 ± 4.15 7.68 ± 3.81 10.39 ± 4.13 15.54 ± 6.29 1495 Propionic acid 6.30 ± 3.99 9.33 ± 6.82 17.02 ± 7.59 23.85 ± 10.49 28.44 ± 10.88 1570 Isobutyric acid 0.56 ± 0.34 0.64 ± 0.20 0.59 ± 0.20 0.89 ± 0.39 0.29 ± 0.09 1597 5-Methylfurfural 0.14 ± 0.09 0.18 ± 0.06 0.20 ± 0.10 0.24 ± 0.08 0.28 ± 0.08 1610 n-Butyric acid 2.40 ± 4.95 0.99 ± 0.52 1.00 ± 0.44 1.28 ± 0.60 1.40 ± 0.38 1659 Ethyl decanoate 0.01 ± 0.01 0.006 ± 0.003 0.006 ± 0.003 0.005 ± 0.003 0.03 ± 0.01 1665 Isovaleric acid 1.03 ± 0.54 1.33 ± 0.52 2.17 ± 0.97 2.02 ± 0.70 0.37 ± 0.09 1707 Diethyl succinate 17.38 ± 8.15 24.61 ± 7.37 28.14 ± 11.88 22.83 ± 8.91 2.06 ± 1.03 1716 n-Valeric acid 0.44 ± 0.22 0.32 ± 0.30 0.24 ± 0.19 0.26 ± 0.21 0.20 ± 0.19 1772 2-Phenethyl acetate 0.16 ± 0.10 0.23 ± 0.16 0.20 ± 0.11 0.12 ± 0.06 0.21 ± 0.15 1863 Hexanoic acid 0.24 ± 0.09 0.16 ± 0.08 0.18 ± 0.06 0.16 ± 0.06 0.47 ± 0.16 1876 Guaiacol 0.21 ± 0.15 0.13 ± 0.04 0.20 ± 0.08 0.14 ± 0.05 0.014 ± 0.01 1909 trans-oak-lactone 0.01 ± 0.003 0.01 ± 0.01 0.01 0.02 ± 0.01 nd^e 1949 2-Phenylethyl alcohol 13.80 ± 4.11 36.72 ± 14.37 67.05 ± 45.20 49.82 ± 19.25 6.89 ± 2.35 1968

cis-oak-lactone 0.08 ± 0.04 0.07 ± 0.05 nd^e nd^e nd^e 2030

o-Cresol 0.03 ± 0.02 0.04 ± 0.03 0.053 ± 0.03 0.07 ± 0.03 0.005 ± 2053 Phenol 0.20 ± 0.10 0.30 ± 0.09 0.29 ± 0.08 0.32 ± 0.10 0.24 ± 0.07 2059 4-Ethylguaiacol 0.013 ± 0.01 0.02 ± 0.01 0.02 ± 0.02 0.015 ± 0.01 0.009 ± 0.01 2090 Octanoic acid 0.92 ± 0.34 0.72 ± 0.45 0.87 ± 0.45 0.97 ± 0.31 3.01 ± 1.30 2097

p-Cresol 0.09 ± 0.07 nd^e nd^e nd^e 0.007 ± 0.01 2134 Eugenol 0.05 ± 0.03 0.05 ± 0.02 0.07 ± 0.04 0.05 ± 0.03 0.008 ± 0.01 2225

Decanoic acid nd^e nd^e nd^e nd^e nd^e 2255^d

2,6-Dimethoxy phenol nd^e nd^e nd^e nd^e nd^e 2274^d

5-(Hydroxymethyl)furfural 56.98 ± 19.37 111 ± 47.31 113 ± 64.23 114 ± 56.37 154 ± 62.81 2528 Vanillin 47.35 ± 27.63 55 ± 31.89 92.83 ± 56.53 34.19 ± 17.73 47.46 ± 23.74 2568

a Average: Average of the detected values only. ^b SD: Standard Deviation of the determined values only. ^c RI: Retention indices from real wine samples and ^d RI from synthetic winecalculated on HP-INNOWax column. ^e nd: not detected.

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Small, but in some cases observable, differences were found in the measured amounts of the analytes in wines, even among those from the same cultivar, producer, and region. For instance in Figure 5.2., the amount of diethyl succinate and phenol measured in four different red wine cultivars is presented for sixteen different producers in South Africa. From this figure it would seem that wine-making procedures, geographical origin, and cultivar plays a more detrimental role in the quality of the wines and not the age since all the wines analyzed were from the 2005 vintage. The data in Figure 5.2. suggests that the method and data generated would prove useful to study the volatile composition of wines and possibility to classify them according to certain criteria such as geographical origin, production technology, or grape variety. This will be the focus of subsequent statistical investigations in future.

Diethyl Succinate

Figure 5.2. Chart representation of a) Diethyl Succinate and b) phenol measured in Pinotage, Shiraz, Cabernet Sauvignon and Merlot wine samples, 16 from each cultivar obtained by HSSE-TD-GC-MS.

(Conditions see text).

NB: CE1 to CE16 = Cellar 1 to Cellar 16 suppliers of the wine samples. Each cellar represents same region but different cultivar.

5.4. Conclusions

In conclusion the developed analytical technique based on stir bar technology was found very sensitive and suitable for the analysis of trace and ultra-trace compounds.

HSSE extraction was very advantageous in reducing the risk of contamination and increasing the lifetime of the PDMS coated stir bar.

The overall results are satisfactory for the analysis of volatile compounds in wine responsible for its aroma achieving low detection and quantification limits. The methodology proposed in this paper allowed us to determine the 37 most important volatile compounds partially responsible for the aroma of wines in a relatively quick

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Although SBSE is a very sensitive technique, PDMS, a non-polar phase, is the only polymer at present adopted as coating of stir bars. This results in poor recoveries of polar compounds with low octanol–water partition coefficients (Ko/w). This was improved by pH adjustment especially for the organic acids. However, a dual-phase twister approach could bring some solution to the limitation of the current stir bar technology by utilizing a material which retains both polar and non-polar compounds.

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