TITRATABLE ACID OF ALCOHOLIC BEVERAGES Total Titratable Acid of Alcoholic Beverages Aim: To find the total titratable acid in wine to determine what packaging has the best preservability and quality through balancing of sugars, acids, and fruit flavours. Introduction Variable geographical, geological, climate and whether are known as “terriors” when discussing factors that influence the ratio between acid and sugar in ripening fruits, creating distinctive features in flavour (Vinepair, n.d.). The fermentation process allows a redox reaction to occur as the yeast reacts with glucose in fruit juice to undergo glycolysis to produce pyruvic acid in anaerobic respiration (Alba- Figure 1 Diagram of Glycolysis Lois et al.,2010). During this fermentation, nicotinamide adenine dinucleotide (NAD) in its oxidation or reduced state NAD+ and NADH respectively. Via acetaldehyde does this redox reaction take place to form either ethanol or carbon dioxide (CO2) (Liu et al., 2017) (Fig 1). Terriors affect the balance between the acid, sugar and fermented alcohol. Generally warmer climates will cause grapes to yield higher sugar concentrations and low acidity, whilst cold climates will produce grapes with greater acidity in their ratio (Puckette, 2015). Both climates can produce good or poor-quality wine. The main difference between low- and highquality wine is the timing of when the grapes are harvested to have the most desirable acid to sugar ratio. Generally, acceptance ratings from consumers decreased with higher acid Figure 2 Graph showing the correlation between acid concentration in wine and consumer concentrations (Fig 2) (Jayasena et al., 2007). acceptance rate “Flavonoids” are polyphenols that are another key component in determining the quality of grape wine. Through phenolics, wine texture and mouthfeel can enhance from its function as metabolites (Gawell et al., 2014). Anthocyanins (pigments), flavanols (UV protectors) and tannins fall under the flavonoid category, each subject to biosynthesis, accumulation and degradation change from high temperatures in particular (Gouot et al, 2019). Whilst pH and titratable acids (TA) both measure acids (titratable acids in molarity [moles/litre]), pH indicates the hydrogens’ ability to disassociate, however TA measures the total amount of disassociated hydrogen protons (Tyl et al., 2017). pH gives a better understanding of microorganism’s ability to grow in foods while the TA indicates the impact of organic acids on foods, and flavour characteristics of wine (The Australian Wine Research Institute, n.d.). Although both measure acids, there is no direct predictability between the two measurements. The ratio between acids and sugars in ripening grapes is crucial as the glucose in sugar ferments into alcohol, and TA contribute to preservability and sensory characteristics of wine (most prevalent in wine are tartaric acid, malic acid, and citric acid) (Rajković et al., 2007). Microbial degradation will be hindered with the presence of acids as high pH will promote the growth of spoiling microbe (Shanker et al., 2021). Acids act as a preservative as pathogens die at a pH below 4.7, however only weak acids “inhibit the growth of the microbe in its undissociated forms at low pH conditions” (Shanker et al., 2021). Although high Figure 4 Diagram of Tannin acidity is not beneficial to the flavour profile of wine, it allows Polymer in Chain wines to be aged which will in turn provide a more pleasurable wine with previously hidden flavours as a result of continued fermentation in anaerobic respiration (Krebiehl, 2018). As wine ages, tannins lose their charge and form into larger chains (Fig 3), falling as sediments to leave a smoother, rounder and more gentle wine, however, if oxidised, the tannins will depolymerize tannin chains, and their surface area will increase (Krebiehl, 2018). The observed Figure 3 Scale Showing how Colour Changed in colour reflects the aging process of wine as tannin Wine Over Time chains grow larger, leaving less anthocyanintannin polymers, allowing colour change to occur as anthocyanin (a flavonoid) functions in changing pigment (Fig 4) (Russan, 2019). The procedure of wine making (Fig 5) further affects the quality and TA present. If the wine is oaked, malic acid is converted to lactic acid which is a softer acid, leading to a higher quality wine (Brittain, 2001). Due to low pH and a weak TA, microbes are not able to grow under this condition, creating a wine that is more preservable. Bottled and cask wine differ in quality as acidity will vary during the shelf-life of cask wine. Due the porous bags which contain the wine, oxygen particles oxidise the wine resulting in an expiration date for cask wine, whilst bottled wine cannot be oxidised when unopened (Johnson, n.d.). Oxygen’s high electronegativity weakens O–H bonds through reduction by Figure 5 Diagram of the Wine Production Process drawing away electrons, increasing the strength of acids (Chemistry Libretexts, 2020). Organic wines further differ in pH, as there is the absence of synthetic chemicals in the grapes when ripening (Honan, 2015). As pesticides are slightly acidic with a “pH between 5.5 and 6.5” (Winfield United, 2015) and herbicides are generally acidic (Bechman, 2016), no artificial acids are introduced to organic grapes. However, as no herbicides are used to alter soil pH, ideal conditions for grapes cannot be created. A controlled soil pH of 5.5 to 6.5 provide the best growing conditions for grapes (Brown, 2013). Furthermore, the growth of weeds draws nutrients from soil and slightly acidify it, leading to the grapes to become more acidic, and in turn an increase in acidity for the wine (WalterPeterson, 2013). When calculating total TA, ratio of different acids is unknown. As 90% of acidity in wine is a combination of tartaric acid and malic acid one of these acids will be necessary during calculations (Joye, 2019). The average percentage of total TA in fruits at early harvest are 56% and 50% respectively whilst 57% and 45% during late harvest, hence, tartaric is the most present TA (Kliewer et al., 1967). As Tartaric is the most present TA in the fruit and wine, calculations are made assuming tartaric acid as the total TA, being expressed as g/L tartaric acid. Sulfur dioxide (SO2 ) and Carbon dioxide ( CO2 ) affect titration after reacting to water through the acidic interference of Sulfuric acid (H₂SO₄) and carbonic acid (H₂CO₃) respectively. The impact of SO2 is negligible due to minimal contribution, however, the presence of CO2 can equate to errors over 1g/L if sample is not degassed (Iland, 2000). Additionally, as CO2 is absorbed by the standard solution of Figure 6 Titration Curve with Carbon Dioxide Interference alkali sodium hydroxide (NaOH), the end point of an acid-base titration changes over time as sodium carbonate is produced, weakening the NaOH with time (Shahorin, 2021). The presence of CO2 in a titration effectively creates a new equivalence point (Fig 6) as the strength of a NaOH standard solution decreases, and acidity increases when water reacts with CO2 to produce H₂CO₃. The total TA of a wine signifies the quality and preservability through the balance between sugars and fruit flavours inside. Therefore, the aim of this investigative is to determine which form of wine is the best quality for consumers. Results Trial 1 Trial 2 Trial 3 Standardisation of NaOH with KHP Initial Volume (mL) Final Volume (mL) 0.600 47.60 0.550 48.20 0.200 47.80 Table 1 Table showing the standardisation of NaOH Titre Value (mL) 47.00 47.65 47.60 Trial 1 Trial 2 Trial 3 Titration of Organic Wine Initial Volume (mL) Final Volume (mL) 0.500 12.20 2.350 14.00 3.000 14.75 Titre Value (mL) 11.70 11.65 11.75 Table 2 Table showing volume to neutralise organic white wine Trial 1 Trial 2 Trial 3 Titration of Bottled Wine Initial Volume (mL) Final Volume (mL) 1.100 12.75 26.00 37.70 37.70 49.40 Titre Value (mL) 11.65 11.70 11.70 Table 3 Table showing volume required to neutralise bottled white wine Trial 1 Trial 2 Trial 3 Titration of Cask Wine Initial Volume (mL) Final Volume (mL) 26.95 40.40 1.600 14.70 29.30 43.00 Titre Value (mL) 13.45 13.10 13.70 Table 4 Table showing volume required to neutralise cask white wine Calculations Calculating the molarity of NaOH KHP moles were found and divided by volume in litres to find concentration using formula: 𝑛 C=𝑣 Moles of KHP used in standardising was then found by multiplying concentration and volume used in litres, giving the formula: n=cxv KHP and NaOH have a 1 : 1 molar ratio, therefore concentration of NaOH can be found with the formula: c= 𝒎 𝒗 where v is in litres, m is moles, and c is concentration in molarity. Calculating the total TA in wine samples Moles of NaOH used is calculated by multiplying the concentration of NaOH and volume used in litres, giving the formula: n=cxv tartaric acid (C4 H6 O6 ) molar ratio to NaOH is 1 : 2, therefore half the moles of NaOH. Concentration is then found using the formula: 𝑛 C=𝑣 Calculating the molarity of NaOH Mass of KHP used: 2.5567 c(KHP) = ( 2.5567 ) 204.22 0.250 c(KHP) = 0.050077M n(KHP) = 0.050077 x 0.04742 n(KHP) = 0.002375 mol Calculating the total titratable acidity of bottled white wine n(NaOH) = 0.095 x 0.01168 n(NaOH) = 0.0011096 1 n(C4 H6 O6 ) = 2 x n(NaOH) n(C4 H6 O6 ) = 0.0005548 mol c(C4 H6 O6) = n(KHP) = n(NaOH) c(NaOH) = 0.002375 0.025 0.0005548 0.025 c(C4 H6 O6) = 0.02219M c(NaOH) = 0.095M (2sf) c(NaOH) = 0.1M (1sf) Calculating the total titratable acidity of organic white wine Calculating the total titratable acidity of cask white wine n(NaOH) = 0.095 x 0.0117 n(NaOH) = 0.095 x 0.01342 n(NaOH) = 0.001115 mol n(NaOH) = 0.0012749 1 1 n(C4 H6 O6 ) = 2 x n(NaOH) n(C4 H6 O6 ) = 2 x n(NaOH) n(C4 H6 O6 ) = 0.00055575 n(C4 H6 O6 ) = 0.000637545 c(C4 H6 O6) = 0.00055575 0.025 c(C4 H6 O6) = 0.02223M c(C4 H6 O6) = 0.000637545 0.025 c(C4 H6 O6) = 0.02550M Figure 7 Calculations showing concentration of total titratable acids in various wines and NaOH standardisation Figure 8 Graph comparing concentration of each wine type Discussion The results of this experiment determined white wine stored in cask packaging had the highest concentration of total TA with 0.02550M (fig 7). Bottled and cask had substantially less with 0.02219M and 0.02223M respectively, bottled having the least total TA (Fig 8). The independent variable for this experiment was the control over the burette. As a result, the dependent variable was when the alcohol would have been neutralised as a reaction to how much volume of a base was added. Other variables which were controlled were the maintenance of clean equipment, a dry pipette bulb, the amount of Potassium hydrogen phthalate (KHP) used in degassing the unknown wine solution, and the volume of the aliquot. These variables were controlled to ensure that each trial had the same conditions. Given the background research, the results corresponded to which wine type would have theoretically held the most and least total TA. Due to the porous bags used in cask wine, oxygen particles weaken O-H bonds, increasing the strength of titratable acids over time, indicating cask wine to yield the highest TA. Due to the lack of controlled growing environments for organic grapes, higher acidity will be present as the soil will contain less nutrients and lose pH, leaving organic wine to have a theoretically higher total TA than bottled wine. The slight difference in TA between bottled and organic can be observed in figure 8. Although results were concurrent to theory, improvements to more reliable and accurate data could have been made. Given more time, additional trials could have been conducted to obtain more accurate results. Furthermore, human error in titrating could be reduced through an automatic titrator to increase the accuracy of burette for better detection of the endpoint. Through better control over volume of standard solution used, calculations would benefit through better data capture. Furthermore, with reliance on technology for quick and accurate use of burette, more trials could be conducted as less time would be spent titrating. Additional human error was made in evaluating the end point of each titration. Colour change from pale yellow to pale pink is subtle and difficult to observe. With the use of green paper underneath the aliquot, subtle colour change was more observable (fig 9), however, the colour change was still too subtle, causing difficulty in finding multiple concurrent titre values. Furthermore, the presence of CO2 affected the ability to gather concurrent results Figure 9 Picture showing the colour of endpoint in titration against green paper as CO2 in the air is absorbed by NaOH to form sodium carbonate, weaking the acid, altering the equivalent point over time as more volume is required to neutralise the TA. As a result of the weaking base, the colour of the solution would revert, making the end point difficult to determine. Different titre values were recorded during the standardisation of NaOH (Table 1), meaning the instability of the solution. As the standard solution was not stable due to the absorption of CO2 , the titration of the wines was made difficult due to a wide range of results from the varying strength of NaOH. Only until three concordant results were found could the total TA be discovered. Better degassing methods could have been used to ensure the absence of CO2 in producing H2 CO2, affecting the acid concentration in the aliquot. To minimise CO2 exposure, the titration should be conducted under a vacuum and the aliquots should be degassed by a strong vacuum or to be heated briefly boiled (Iland, 2000). De-ionised water should also be freshly boiled and cooled. With these conditions, no CO2 would be able to affect the endpoint of the titration, resulting in more accurate and concurrent results. In determining the total TA, the volume of each acid in the aliquot should be known. Tartaric acid was used to calculate the total TA due to it being the most predominant TA in white wine. Finding the true total TA, the moles of each present need to be known to find the accurate g/L of TA. Currently tartaric acid is used to give a general understanding of the approximate total titratable acid present. As bottled wine displayed the least TA, it is the best quality wine for consumers as its ability for preservability is the best with the weakest acids, does not oxidise without being opened, and as a result tannins would have the least surface area. Furthermore, with low TA, the acid to sugar ratio is symbolic of quality wine, exhibiting terroir with grapes ripened in warmer climates. Conclusion Through the experiment, the best quality wine is found to be in bottled wine. Organic displays better wine quality than cask wine due to cask wine’s high total TA. Additionally, the low TA in bottled wine displays optimal acid : sugar ratio compared to other forms of white wine.. References Alba-Lois, L. and Segal-Kischinevzky, C., 2010. Yeast, Fermentation, Beer, Wine | Learn Science at Scitable. [online] Nature.com. Available at: <https://www.nature.com/scitable/topicpage/yeast-fermentation-and-the-making-ofbeer-14372813/> [Accessed 23 April 2022]. o Bechman, T., 2016. Why you need to know about pH when spraying herbicides. [online] Farm Progress. Available at: <https://www.farmprogress.com/story-why-need-knowph-spraying-herbicides-9-136707> [Accessed 21 April 2022]. o This source explains how oxidation increases the strength of acids through the weakening of O-H bonds. Compound Interest. 2014. The Key Chemicals in Red Wine – Colour, Flavour, and Potential Health Benefits. [online] Available at: <https://www.compoundchem.com/2014/05/28/redwinechemicals/> [Accessed 26 April 2022]. o Had a titration curve with how carbon dioxide created new equivalence point. Chemistry LibreTexts. 2020. 16.10: Acid Strength and Molecular Structure. [online] Available at: <https://chem.libretexts.org/Bookshelves/General_Chemistry/Map%3A_A_Molecular_A pproach_(Tro)/16%3A_Acids_and_Bases/16.10%3A_Acid_Strength_and_Molecular_Stru cture#:~:text=Any%20inductive%20effect%20that%20withdraws,the%20strength%2 0of%20the%20acid.> [Accessed 25 April 2022]. o This source explains the best pH conditions for the ideal grapes to grow. Cark, J., 2002. pH curves (titration curves). [online] Chemguide.co.uk. Available at: <https://www.chemguide.co.uk/physical/acidbaseeqia/phcurves.html> [Accessed 25 April 2022]. o Explains how softer acids makes wines more palatable. It further explains how malic acid turns into lactic acid. Brown, D., 2013. Soil sampling vineyards and guidelines for interpreting the soil test results. [online] MSU Extension. Available at: <https://www.canr.msu.edu/news/soil_sampling_vineyards_and_guidelines_for_interpr eting_the_soil_test_resul> [Accessed 21 April 2022]. o Explains the chemistry of herbicides and how they are usually acidic, affecting the acidity of non-organic grapes. Brittain, H., 2001. Analytical profiles of drug substances and excipients. 28th ed. San Diego: Academic Press, pp.153-195. o Explains glycolysis in fermentation and the role of pyruvic acid. Had a graph of tannin chains of how the chain together and polymerise. Gawel, R., Godden, P., Williamson, P., Francis, L., Smith, P., Waters, L., Herderich, M. and Johnson, D., 2014. Influence of phenolics on white wine quality and style. Wine & Viticulture Journal, [online] 29(3), pp.34-36. Available at: <https://issuu.com/provincialpressgroup/docs/wvj_v29n3-v2_lr> [Accessed 23 April 2022]. o Gouot, J., Smith, J., Holzapfel, B., Walker, A. and Barril, C., 2019. Grape berry flavonoids: a review of their biochemical responses to high and extreme high temperatures. [online] National Library of Medicine. Available at: <https://pubmed.ncbi.nlm.nih.gov/30388247/> [Accessed 24 April 2022]. o Describes why cask wine has a shelf life as the bags that hold the wine are porous and allow oxygen particles to oxidise the alcohol. Joye, I., 2019. Encyclopedia of Food Chemistry. 1st ed. [ebook] Guelph: Elsevier, pp.1-9. Available at: <https://doi.org/10.1016/B978-0-08-100596-5.21582-5> [Accessed 25 April 2022]. o Demonstrates consumer approval rating to a change of ratio between acidity concentration and sugar concentration. Johnson, M., n.d. 6 Must-Try Seltzers For The Summer. [online] Spoon University. Available at: <https://spoonuniversity.com/lifestyle/five-must-try-seltzers-for-thesummer> [Accessed 23 April 2022]. o Provides information on how to correctly titrate with external influences such as the interference of carbon dioxide. Jayasena, V. and Cameron, I., 2007. °BRIX/ACID RATIO AS A PREDICTOR OF CONSUMER ACCEPTABILITY OF CRIMSON SEEDLESS TABLE GRAPES. [ebook] Perth: Department of Agriculture and Food Western Australia, p.11. Available at: <https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1745-4557.2008.00231.x> [Accessed 27 April 2022]. o Explains how organic grapes differ in acidity due to the lack of pesticides and herbicides. Iland, P., 2000. Techniques for chemical analysis and quality monitoring during winemaking. 1st ed. Campbelltown: Patrick Iland Wine Promotions. o Explains flavanols and how they are affected by temperature. Honan, D., 2015. Why you should be drinking organic wine and where to find it. [online] the Guardian. Available at: <https://www.theguardian.com/lifeandstyle/australia-foodblog/2015/feb/07/why-you-should-be-drinking-organic-wine-and-where-to-find-it> [Accessed 23 April 2022]. o Explains flavanols and how phenols impact the quality and characteristics of wine. This source explains how tartaric and malic acid make up most acids in alcohols. Kliewer, M., Howarth, L. and Omori, M., 1967. Concentrations of Tartaric Acid and Malic Acids and Their Salts in Vitis Vinifera Grapes. American Journal Enology and Viticulture, [online] 18(1), pp.42 - 54. Available at: <https://www.ajevonline.org/content/18/1/42#:~:text=For%20all%20varieties%2C %20the%20average,45%25%20at%20the%20late%20harvest.> [Accessed 25 April 2022]. o Gives percentages of which acid is most dominant in grapes. Tartaric acid and malic are the most present, however, tartaric acid has a greater percentage. Krebiehl, A., 2022. What Really Happens as Wine Ages? | Wine Enthusiast. [online] Wine Enthusiast. Available at: <https://www.winemag.com/2018/10/09/what-happenswine-ages/> [Accessed 23 April 2022]. o Liu, C., Qin, J. and Lin, Y., 2017. Fermentation and Redox Potential. Fermentation Processes, [online] Available at: <https://www.intechopen.com/chapters/51634> [Accessed 24 April 2022]. o Report which goes into detail the titratable acids present in wine and how they affect the preservability and characteristics of it. Russan, A., 2019. The Science of Color in Wine | SevenFifty Daily. [online] SevenFifty Daily. Available at: <https://daily.sevenfifty.com/the-science-of-color-inwine/#:~:text=Tannin%2Danthocyanin%20polymerization%20in%20wine,result%20i n%20a%20softer%20mouthfeel.> [Accessed 26 April 2022]. o Describes why different acidity affects characteristics of the wine Rajkovic, M., Novakovic, I. and Petrovic, A., 2007. Determination of titratable acidity in white wine. Journal of Agricultural Sciences, Belgrade, [online] 52(2), pp.169-184. Available at: <http://www.doiserbia.nb.rs/img/doi/1450-8109/2007/145081090702169R.pdf#:~:text=Wines%2C%20as%20a%20rule%2C%20contain,or%20ar gol)%20during%20alcohol%20fermentation.> [Accessed 22 April 2022]. o Gives the process of how white wine is made from grapes. Picture from this site was used that gave the process. Puckette, M., 2015. Understanding Acidity in Wine | Wine Folly. [online] Wine Folly. Available at: <https://winefolly.com/deep-dive/understanding-acidity-in-wine/> [Accessed 23 April 2022]. o Says how cool climate and warm climates affect the sugar-acid ratio within grapes. Puckette, M., 2013. How White Wine is Made From Grapes to Glass | Wine Folly. [online] Wine Folly. Available at: <https://winefolly.com/deep-dive/how-is-white-wine-made/> [Accessed 25 April 2022]. o Explains how NADH and NAD+ affects the process of fermentation, undergoing redox reaction. Mowery, L., 2018. The Real Difference Between Cool-Climate and Warm-Climate Wine | Wine Enthusiast. [online] Wine Enthusiast. Available at: <https://www.winemag.com/2018/05/01/cool-vs-warm-climate-wine/> [Accessed 24 April 2022]. o Explains how aging of the wine changes the flavour characteristics. Article that explains the change of colour in wine after aging. Shahorin, W., 2021. How does carbon dioxide affect titration?. [online] Everythingwhat.com. Available at: <https://everythingwhat.com/how-does-carbondioxide-affect-titration> [Accessed 23 April 2022]. o Website that details how carbon dioxide affects titration with NaOH as it weakens the base, and turn solutions more acidic when reacted with water. Shankar, V., Mahboob, S., Al-Ghanim, K., Ahmed, Z., Al-Mulhm, N. and Govindarajan, M., 2021. A review on microbial degradation of drinks and infectious diseases: A perspective of human well-being and capabilities. Journal of King Saud University Science, [online] 33(2), p.101293. Available at: <https://www.sciencedirect.com/science/article/pii/S1018364720304067> [Accessed 23 April 2022]. o The Australian Wine Research Institute. n.d. Acidity and pH - The Australian Wine Research Institute. [online] Available at: <https://www.awri.com.au/industry_support/winemaking_resources/frequently_asked _questions/acidity_and_ph/#:~:text=Precipitation%20of%20potassium%20bitartrate% 20is,titratable%20acidity%20(TA)%20decreases.> [Accessed 23 April 2022]. o Website that explains what terrior are and its relevance to wine. Walter-Peterson, H., 2013. How Soil pH Influences Grapes. [online] Growingproduce.com. Available at: <https://www.growingproduce.com/fruits/commentary-how-soil-phinfluences-grapes/> [Accessed 23 April 2022]. o Report that goes further into detail the difference between pH and titratable acidity. This being the ability to measure the ability of hydrogen ions to disassociate and the concentration of disassociated hydrogen ions. VinePair. n.d. Learn About Terroir | Understanding Terroir | Wine 101. [online] Available at: <https://vinepair.com/wine-101/terroir-wine-guide/> [Accessed 26 April 2022]. o Explains the difference between pH and titratable acidity when measuring acids in alcohol. Tyl, C. and Sadler, G., 2017. pH and Titratable Acidity. 1st ed. [ebook] Cham: © 2017 Springer International Publishing, pp.389 - 406. Available at: <https://link.springer.com/chapter/10.1007/978-3-319-45776-5_22#citeas> [Accessed 25 April 2022]. o Report that explains the perfect conditions to prohibit microbial and pathogen growth, being a combination of low pH and weak TA. Details how the pH of soil affects the acid to sugar ratio within ripening grapes. Winfieldunited.com. 2015. Pesticide Performance and pH Levels. [online] Available at: <https://www.winfieldunited.com/news-and-insights/pesticide-performance-and-phlevels> [Accessed 24 April 2022]. o Gives an understanding of the pH of majority of pesticides. This then influences the acidity within the growing grapes.